bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualizing multiscale models of the nephron
Authors: Terkildsen Jonna , Nickerson David , Hamilton Kirk , Hunter Peter
Published: June 2021

We present the development of a tool which provides users with the ability to visualize and interact with multiscale models of the nephron – from the scale of models of membrane bound proteins, to that of an individual nephron.

A 1‐D finite element model of the nephron has been created and is used for both visualization and modeling of the tubule transport. Mathematical models of nephron segments (for example, Weinstein et al., Am. J. Physiol. 292:F1164‐F1181, 2007 for the proximal tubule) are embedded in the finite element model. At the cellular level these segment models utilize models encoded in CellML (www.cellml.org) to describe cellular transport kinetics.

A user interface has been developed which allows the visualization and interaction with the multiscale nephron models and simulation results. The zinc extension to Firefox (http://www.cmiss.org/cmgui/zinc/) is used to provide an interactive 3‐D view of the model(s). This model viewer is embedded in a web page which dynamically presents content based on user input. For example, when viewing the whole nephron model the user might be presented with information on the various embedded segment models as they select them in the 3‐D model view. Similarly, the user might choose to focus the model viewer on a cellular model in a particular segment in order to view the various membrane transport proteins. Selecting a specific protein might present the user with a full reference description of the mathematical model governing the behavior of that protein (Nickerson et al., Bioinformatics 24:1112‐1114, 2008).
Towards a global sensitivity analysis of the Guyton model of blood pressure regulation.
Authors: Bazin Jérôme , Grosse Thibault , Thomas S. Randall
Published: June 2021

We present a partial sensitivity analysis of the Guyton model of blood pressure regulation. Starting from a steady state, 190 selected model parameters were systematically perturbed one at a time, and the effect on the 286 selected model variables was evaluated at 1 minute, 5 minutes, 1 hour, 1 day, and 1 week after the perturbation.

Results

parameters of a given sub‐module did not preferentially affect variables from the same sub‐module. Rather, most variables are relatively insensitive to small parameter changes, in confirmation of the system's robustness. However, those variables that are sensitive show sensitivity to many parameters. Details will be given in the presentation.

Conclusions

This one‐at‐a‐time parameter study is a useful start towards a comprehensive study, but it will be more physiologically interesting to study sequential changes of several parameters. This will require a sensible strategy to avoid combinatorial explosion of CPU time.
Epigenetic modification of Huntingtin associated protein‐1 by bioactive natural components as a strategy for the prevention and treatment of breast cancer
Authors: Zhou Jin‐Rong , Abdolmaleky Hamid , Eskandari Mohamad , Blackburn George
Published: June 2021

The objectives of this study were to determine if Huntingtin associated protein‐1 (HAP1) could serve as a functional target for breast cancer (BRCA) and if bioactive natural components that modulate HAP1 expression and function may have preventive and/or therapeutic activities against BRCA. Searching for novel targets for effective prevention and therapy remains the to priority in BRCA research. The role of HAP in BRCA has not been investigated. The DNA promoter of HAP1 gene has CpG islands, suggesting that HAP1 expression could be epigenetically modified. Our in vitro studies showed that, compared with the normal breast epithelial cells, human BRCA cell lines had dramatically reduced HAP1 gene expression associated with hypermethylation of its DNA promoter. The results were also confirmed in human samples of cancerous and normal breast tissues. Gain‐of‐function assays showed that overexpression of HAP1 inhibited growth rates of BRCA cell lines. Several bioactive dietary and botanical components inhibited the growth of BRCA cells associated with up‐regulation of HAP1 gene expression and DNA promoter hypomethylation, with tanshinone IIA the most potent component. These results suggest that HAP1 could serve as a functional target for BRCA, and epigenetic modification of HAP1 expression may provide a novel approach to identify bioactive dietary and natural components for prevention and treatment of BRCA.
P2X7 RECEPTOR PLAYS A CRUCIAL ROLE IN LPS‐MEDIATED ACUTE LUNG INJURY
Authors: Mishra Amarjit , Weng Tingting , Breshears Melanie , Chintagari Narendranath R , LIU LIN
Published: June 2021

Purinergic P2X7 receptor is an ATP‐gated ion channel and participates in inflammation by regulating IL‐1β processing and release. Recent studies have identified the pathophysiological importance of P2X7 receptor in immune and neuroinflammatory diseases. However, the role of P2X7 receptor in acute lung injury is still unclear. In the present study using a two‐hit lung injury model of low dose endotoxin exposure and moderate tidal volume mechanical ventilation for 3 hours, we compared the early inflammatory response between wild‐type and P2X7−/−mice. There was a significant reduction in total bronchoalveolar lavage cell counts and neutrophil infiltration in lung tissue and lavage in P2X7−/− mice compared to wild‐type. Furthermore, myeloperoxidase from P2X7−/− mice lung homogenate was also reduced. The release of proinflammatory cytokine IL‐1β and IL‐6, but not TNF‐α from P2X7−/− mice showed a marked reduction. In parallel, transcripts of IL‐1β, IL‐6, and MCP‐1 from P2X7−/− mice failed to respond to challenge. However, TNF‐α, KC, and IL‐10 shows a similar expression pattern between wild‐type and P2X7−/−mice. Moreover, P2X7−/− mice exhibited less responsiveness to alveolar–capillary leakage, as measured by reduced bronchoalveolar lavage protein accumulation and Evans blue dye extravasion. Taken together our results support that P2X7 receptor facilitates IL‐1β and IL‐6 release and is involved in the pathogenesis of acute lung injury.
The Salmonella effector AvrA mediates bacterial intracellular survival during infection
Authors: Wu Huixia , Jones Rheinallt , Neish Andrew
Published: June 2021

The enteric pathogen Salmonella typhimurium secretes the preformed effector AvrA into host cells. This acetyltransferase has been shown to suppress intestinal immune and apoptotic responses by inhibition of JNK MAPK. To study the role of AvrA in natural enteric infection, we used a mouse model to compare wild type Salmonella to an isogenic AvrA null mutant. Salmonella lacking AvrA induced increased neutrophil infiltration in the gut, more intense systemic cytokine responses, and higher apoptosis in epithelial cells. Mice infected with an AvrA‐ mutant also showed higher bacterial burden in liver, spleen, and mesenteric lymph nodes. Furthermore, the mice exhibited more severe apoptosis in macrophages within those tissues. Consistently, in vitro primary epithelial cells and mouse bone marrow derived macrophages infected with AvrA‐ mutant Salmonella also showed higher apoptosis relative to wild type bacteria. To study the mechanisms involved, recombinant adenoviruses expressing AvrA were constructed. AvrA transduced into epithelial cells and macrophages protected the cells from an external proapoptotic signal. In conclusion, the results suggest AvrA suppresses apoptosis in infected host cells, which may allow for establishment of a stable niche typical of intracellular pathogens.
Undergraduate SMART Teams: Exposing Students to the Process of Science through Physical Modeling
Authors: Herman Tim , Franzen Margaret , Colton Shannon , O'Neal Marvin
Published: June 2021

A common problem faced by both large and small undergraduate institutions is the relatively small number of research experiences available for undergraduate students. As one solution to this problem, we are piloting a new undergraduate course, PREPARE, in which teams of students work with an active research lab to design and build, using 3D‐printing technology, a physical model of a protein that is being investigated in the lab. The course is divided into three phases. In the Training Phase, students learn to read primary literature, give a journal club, and construct a physical model of a simple protein. In the Design Phase, teams of 3–4 students work closely with a research lab to understand the research project and to create a physical model that tells the molecular story of a protein involved in the work. In the Presentation Phase, teams “publish” their work as a Proteopedia page and as a poster for a scientific meeting. This new course is designed to provide sophomore undergraduates with an authentic research experience and to develop criteria that faculty can use to select qualified and motivated students who wish to gain experience in their research lab. This work was supported by an NSF‐CCLI award to TH.
Integrating Comparative & Experimental Methods to Better Understand the Functional Morphology of Primate Hand Bones
Authors: Patel Biren Ashok
Published: June 2021

The present study examined the functional morphology of primate hand bones in order to identify biomechanically relevant osteological correlates of habitually terrestrial digitigrade behaviors. As predicted, digitigrade species have relatively shorter phalanges and metacarpals compared to their arboreal counterparts; shorter hand bones help counteract potentially higher bending moments resulting from higher GRFs during terrestrial locomotion. In contrast, relative robusticity of these bones were not different between groups, and analyses indicate that metacarpal shape weakly discriminates primates based on hand posture behavior. Subsequent in vivo kinematic and kinetic studies of primate locomotion were conducted to lend insight on these observed morphological differences and similarities. It was found that digitigrade hand postures were used only at slow speeds when GRFs are low. Thus, adopting an extended digitigrade hand posture helps increase effective limb length in animals with short hand bones. At faster speeds with higher GRFs, the hands transitioned to a more palmigrade posture and analysis of dynamic pressure demonstrated that increasing palmigrady at faster speeds increased palmar contact area and attenuated pressure in the hand during locomotion. Accordingly, it is likely that stresses are relatively low in fragile hand bones irrespective of substrate used, thereby not requiring terrestrial digitigrade species to have more robust hand bones than arboreal species. The results of this study demonstrate the utility of combining comparative and experimental methods to better understand functional morphology.
Shouldering the burdens of locomotion & posture: glenohumeral joint structure in strepsirrhines.
Authors: Burrows Anne M , Wright‐Fitzgerald Adrian S
Published: June 2021

The present study compares soft‐tissue morphology of the shoulder joint (acromioclavicular, coracoclavicular, and glenohumeral joints) among broad phylogenetic, locomotor, and postural behavior ranges in prosimian primates. Adult specimens of Galago moholi (a vertical clinger and leaper), Cheirogaleus medius (an arboreal quadruped), Eulemur macaco (an arboreal quadruped that also frequently engages in suspensory behavior), and Tarsius syrichta (a vertical clinger and leaper) were dissected. The rotator cuff muscles were also dissected and weighed among the species. Results showed that muscle mass of individual components of the rotator cuff musculature do appear to be adaptive. Two soft‐tissue components of the glenohumeral joint, but not the acromioclavicular and coracoclavicular joints, also were adaptive. The quadrupedal species, C. medius and E. macaco, both had glenohumeral ligaments and E. macaco, which also uses suspensory postures, had a relatively deeper articular surface for the humerus due to the shape of a deep, “cup‐like” glenoid labrum. Additionally, the present study noted a lack of a teres minor muscle in G. moholi, C. medius, and E. macaco despite previous studies describing them. A relatively robust teres minor muscle was found in T. syrichta. Overall, these results show that soft‐tissue joint morphology itself may be as adaptive as osseous morphology.
Stereo photogrammetric characterization of wishboning strain in the colobine mandibular symphysis
Authors: Daegling David John , Bucinell Ronald , McGraw William Scott , Rapoff Andrew Jordan
Published: June 2021

The masticatory loading regime of lateral transverse bending (wishboning) is hypothesized to be instrumental in the evolution of symphyseal form among primates. The biomechanics of wishboning have largely been inferred by assuming that the mandible behaves as a curved beam under this load; however, the characterization of stress and strain in the anthropoid symphysis has been interpretively challenging. This is due, in part, to both limitations of sampling strain in an in vivo context and the incongruence of beam theory assumptions on the one hand and the anatomical complexity of mandibular morphology on the other.

Utilizing stereo photogrammetry, we employ an in vitro approach to characterize the strain field in a sample of colobine mandibles under simulated wishboning loads in order to assess the utility of idealized curved beam models for characterizing strain gradients in symphyseal bone. This full‐field method also provides an opportunity to evaluate the effects of morphological variation on the distribution of wishboning strains.

Conventional theory of curved beams suggest that colobine mandibles should exhibit reduced disparity of labial and lingual stresses relative to papionin primates given differences in overall mandibular architecture. This prediction is borne out by our analysis: whereas macaques experience lingual:labial strain disparities of 3.5:1, the colobine mandibles exhibit ratios on the order of 2‐3:1. Despite the fact that wishboning loads represent a case of asymmetric bending, details of the wishboning strain field do not conform to expected stress distribution under this model.
LEF/TCFs and Transcription Regulation
Authors: Waterman Marian L. , Najdi Rani , Hoverter Nate , Pate Kira T. , Marsh J. Larry
Published: June 2021

LEF/TCF transcription factors are nuclear mediators of beta‐catenin‐dependent Wnt signaling. While all LEF/TCFs direct Wnt signals to target genes for regulation, mammalian members of this family also exhibit differences in DNA sequence recognition, dominant negative actions and nuclear/cytoplasmic shuttling. These non‐redundant activities modify the strength and specificity of Wnt signals in the nucleus. Alternative splicing and promoter usage drive some of these non‐redundancies, but the nuclear/cytoplasmic shuttling activity derives from a signal transduction cascade first defined in C. elegans for guiding differential cell fate choices during development. This pathway can be triggered by specific Wnt ligands or calcium calmodulin kinase II. In addition, it functions as a Wnt signaling loop as it can trigger the release of Wnt ligands from cells. The biological consequences of this pathway will be discussed in relation to the expression patterns of LEF/TCFs in development and disease.
The sleepy Neanderthal hypothesis: Relationships among craniofacial form, obstructive sleep apnea and sleep deprivation.
Authors: Laitman B.M. , Pagano A.S. , Reidenberg J.S. , Marquez S. , Monge J. , Laitman J.T.
Published: June 2021

Reconstructions of the Neanderthal upper respiratory tract‐including nasal, pharyngeal, lingual and laryngeal components‐suggest that this region may have differed considerably from that in living humans. Features, such as a decreased oropharynx, or more of the tongue in the oral cavity, have been found in patients with Obstructive Sleep Apnea (OSA), a neuro‐mechanical disturbance of air flow that disrupts proper respiration during sleep and causes disturbed and limited sleep. Here we review current OSA research in comparison with interpretations of Neanderthal anatomy and hypothesize that should Neanderthals indeed have demonstrated an anatomical template conducive to OSA, they may have suffered from this with greater frequency than that found in living humans with concomitant sleep disturbances. Due to Neanderthal's greater reliance on nasal breathing‐as evidenced by uniquely derived features of their nasal complex‐the possibility of increased nasal infections may have put additional stress on their oral air route. Given the above, we raise the question: Could a contributing factor to the Neanderthal's demise be sleep deprivation due to respiratory disturbance?
Assessing millennial student behaviors
Authors: DiLullo Camille , Kriebel Richard M
Published: June 2021

The millennial/net generation student cohort demonstrates behavioral traits which have generated innovative instruction and assessment strategies. Following a 1999 report that sited inappropriate behaviors as a contributing factor for an increase in medical errors, medical educators were prompted to enact an educational strategy for the clinical setting based on professional core competencies which included professional behavior as well as medical knowledge. It is hypothesized that for optimal student success in core competency assessments, this strategy must be modified and extended into the early part of medical training. A process has been devised to guide students in determining whether individual behaviors manifest in college which are related to professional core competencies meet those required at the graduate and professional levels. The process includes a novel student self‐assessment tool that addresses behaviors related to aspects of commitment, professionalism, service, quality of life issues and learning among others. First year medical students were asked to complete the self‐assessment at the beginning of training. A portion of the data collected regarding learning reveals that 64% of respondents reported routinely reading part or all of an assigned text while only 25% retrieve information in an examination setting by recalling or visualizing information they had read.
The potential role for Interferon regulatory factor 6 in keratinocyte adhesion and/or migration
Authors: Olson Leah C , Schutte Brian C , Murray Jeffrey C , Dunnwald Martine
Published: June 2021

Mutations in Interferon Regulatory Factor (IRF6) cause two orofacial clefting syndromes, Van der Woude (VWS) and Popliteal Pterygium (PPS). In addition to an oral defect, recent data show that VWS patients are more likely to have wound complications after corrective cleft surgery than patients with isolated cleft. Thus we hypothesize that Irf6 is critical to wound closure. To test this, we used keratinocytes from Irf6 null mice. We created in vitro scratches and observed a significant delay in scratch closure. To determine the cause of delay, we examined the cellular structure of Irf6−/− keratinocytes by staining for filamentous actin and found that these cells have more stress fibers. In addition, we examined cellular adhesion by staining Irf6−/− keratinocytes for E‐cadherin and vinculin. We found that these cells form junctions, but cell‐cell contacts appear in an abnormal pattern, and there appear to be more focal adhesions. Finally, we analyzed proliferation of Irf6−/− keratinocytes and found no deficiency. Together, our data suggests Irf6−/− keratinocytes exhibit characteristics of less motile cells. Thus, we speculate that the defect in Irf6−/− keratinocyte scratch closure is due to aberrant migration and/or adhesion, and that cellular proliferation is not inhibitory to closure. Broadly, these data suggest that Irf6 is necessary for events critical to proper cutaneous wound healing.

Grant Funding Source: NIH‐NIAMS AR 055313
Placental DHA and ARA are negatively associated with body burden of polychlorinated dibenzofurans (PCDFs) in pregnant women
Authors: Huang Meng‐Chuan , Sun Pei‐Yi , Wang Shu‐Li
Published: June 2021

N‐3 and N‐6 polyunsaturates, DHA (docosahexaenoic acid, 22:6n‐3) and ARA (arachidonic acid, C20:4n‐6), are important in perinatal development. In contrast, increased body burden of polychlorinated dibenzo‐p‐dioxins (PCDD/Fs) or polychlorinated dibenzofurans (PCBs) lead to impaired neurological development. Because of similar chemical properties and origins of food sources, critical delivery of DHA and ARA to the fetus may also contribute to increased exposures to lipophilic organo‐chlorines. This study aimed to examine relationships between placental PCDD/Fs and PCBs toxic equivalent (TEQ) and DHA/ARA levels in 34 pregnant women from Taiwan. Trends of decreases in DHA (p=0.025) were observed as PCDF TEQ increased. Similarly, placental levels of ARA were reduced (p=0.020) as PCDF TEQ levels increased. In contrasts, DHA and ARA did not correlate with TEQ levels of PCDD or PCBs. After adjusting for confounders, increase in one PCDF TEQ were negatively correlated with decreases in 0.312% (wt%) of placental DHA (beta=−0.312, p=0.027) and 1.021% (wt%) of ARA (beta=−1.021, p=0.025). Thus, increases in body burden of PCDFs may alter placental DHA and ARA status. Mechanisms regarding placental DHA and ARA transfer which may be interfered by dioxin exposure warrant further investigation.
Maternal Vitamin D Status and Child Tuberculosis, Anemia, and Morbidity in Tanzania
Authors: Finkelstein Julia L , Mehta Saurabh , Manji Karim P. , Duggan Christopher P. , Mugusi Ferdinand M. , Spiegelman Donna , Msamanga Gernard I. , Fawzi Wafaie W.
Published: June 2021

Objective

To examine the association of maternal vitamin D status with child health outcomes.

Methods

Vitamin D status [serum 25(OH)D] was determined in 884 HIV‐infected pregnant Tanzanian women at enrolment in a trial of vitamins. Children born to these women were followed‐up until 24 months of age and morbidity was recorded. Tuberculosis (TB) was diagnosed via chart review. Proportional hazards models were used to assess the associations of outcomes with maternal vitamin D status.

Results

Children born to women with low vitamin D (<32ng/mL) levels were 1.58 times more likely to develop pulmonary TB (95%CI: 1.01–2.47), after adjusting for age, HIV stage, CD4 count, and regimen. Low vitamin D status was associated with 1.21 times greater risk of cough (95%CI: 1.05–1.40). No significant associations were noted for diarrhea or other respiratory symptoms. For anemia outcomes, no association was observed with low vitamin D status. However, in quintile analyses, children born to women in the second quintile had lower risk of severe anemia (Hb<8.5g/dL) (RR: 0.64; 95%CI: 0.45–0.92) and severe hypochromic microcytosis (RR: 0.34; 95%CI: 0.16–0.71), compared to the lowest quintile.

Conclusion

Vitamin D status may affect risk of child outcomes, such as tuberculosis and anemia. Further research is warranted to examine the role of optimizing vitamin D status in reducing morbidities in children born to HIV‐infected women.

Grant Funding Source: NICHD R01 32257
Interactions of ferroportin with hephaestin on the basolateral membrane of human intestinal cells
Authors: Kim Eun‐Young , Ham Soo‐Kyung , Becker Erin , Han Okhee
Published: June 2021

To investigate interactions between ferroportin (FPN) an iron exporter and hephaestin (Heph) a copper‐bound ferroxidase, we performed experiments using fluorescence energy transfer (FRET). We prepared appropriate fluorescent probes by attaching cyan fluorescent protein (CFP) to the N‐terminus of Heph and yellow fluorescent protein (YFP) to the N‐terminus of FPN. In cells stably expressing Heph‐CFP, we first assessed CFP fluorescence, pixel by pixel. In cells stably expressing both Heph‐CFP and FPN‐YFP, we then reassessed fluorescence of each pixel at the CFP donor emission wavelength. An YFP‐induced decrease in CFP emission suggests that the two proteins are associated. We found that an FPN‐YFP induced the decrease of Heph‐CFP emission suggesting the association of these two proteins on the basolateral membrane of human intestinal Caco‐2 cells. Our FRET experiments show that the distance of FPN‐YFP from Heph‐CFP is ~ 5 nm. Our FRET studies also indicate that neither FPN nor Heph is associated with TfR1 on the basolateral membrane of the enterocyte, although we showed the colocalization of these two proteins with TfR1 on the basolateral membrane of the enterocyte.
Effects of leucine and calcitriol on monocyte‐vascular endothelial cell adhesion
Authors: Curry Benjamin , Biggerstaff John , Zemel Michael B.
Published: June 2021

We previously demonstrated that calcitriol and leucine modulate adipocyte and macrophage cytokine production. Since several of the affected cytokines may modulate vascular infiltration by monocytes, we have tested the hypothesis that calcium (via calcitriol suppression) and leucine attenuate vascular infiltration. Adipocytes were treated with vehicle, calcitriol (to mimic calcium deprivation), leucine or both for 48 hr and conditioned medium (CM) collected and incubated with fluorescently labeled U937 monocytic cells perfused across monolayers of EA hy.926 endothelial cells; adherence was then quantified by microscopy and image analysis. Results are presented as a Monocyte:Endothelial cell ratio (M:E). Calcitriol (10 nM) increased the M:E from 0.088+0.048 to 0.201+0.112 (228% increase, p = 0.0001). Conversely, leucine (0.5 mM) markedly decreased the M:E to 0.013+0.011 (p = 0.001); moreover, addition of leucine CM to the calcitriol CM treatment abolished the effects of calcitriol. A parallel experiment using flow cytometry of CD11b expression on U937 cells under parallel conditions showed an identical trend to the microscopy data. These data show that monocyte adhesion to endothelial cells is decreased in the presence of leucine and increased by calcitriol, suggesting that foods rich in calcium and leucine may be beneficial in reducing monocyte adhesion. Supported by the National Dairy Council.
Specific starch digestion of maize alpha‐limit dextrins by recombinant mucosal glucosidase enzymes
Authors: Lin Amy Hui‐Mei , Nichols Buford L , Quezada‐Calvillo Roberto , Rose David R , Sim Lyann , Hamaker Bruce R
Published: June 2021

Starch digestion requires two luminal enzymes, salivary and pancreatic alpha‐amylase (AMY), and four small intestinal mucosal enzyme activities from the N‐ and C‐terminals of maltase‐glucoamylase (MGAM) and sucrose‐isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose but dramatically amplifies α‐glucogenesis by producing α‐limit dextrins (LDx) as favored substrates for the mucosal enzymes.

Aim

to study substrate specificity of α‐glucogenesis by four recombinant MGAM and SI subunits.

Methods

Recombinant α‐glucosidase domains were produced in drosophila (N‐) or baculovirus (C‐) cell lines and purified by histags. LDx was produced with AMY from normal maize and three mutant cultivars with different amylopectin structures: normal, waxy, ae waxy and du waxy. Glucose released in vitro was measured by GOPOD.

Results

N‐MGAM subunit releases glucose from shortest linear oligomers while the other three subunits digest longer chains or branched molecules.

Conclusions

This is the first time that the individual α‐glucogenic activities of all four mucosal enzyme subunits have been tested. The individual α‐glucosidase subunits had different activities for each cultivar LDx substrate and selective affinities for LDx from different cultivars. Combinations of α‐glucosidase subunits thus provide a broader spectrum of α‐glucogenic activities from structurally varied starch LDx.
An investigation into the profile and dynamics of neutrophil transendothelial cell migration (TEM) using high resolution in vivo real‐time confocal imaging
Authors: Woodfin Abigail , Voisin Mathieu‐Benoit , Colom Bartomeu , Nourshargh Sussan
Published: June 2021

A high resolution real‐time 3D imaging technique was established for analyzing the characteristics and dynamics of neutrophil transmigration in vivo. Our model involves rapid confocal microscopy imaging of the cremaster muscle of lys‐EGFP mice (which exhibit EGFP+‐neutrophils) in conjunction with fluorescently labeled endothelial cell (EC) junctions using an anti‐PECAM‐1 mAb. With this method we have examined the profile and dynamics of neutrophil TEM as elicited by several stimuli (eg IL‐1β, FMLP and ischemia/reperfusion injury). In all reactions the majority of the TEM events occurred via the paracellular route where the formation of EC junctional pores at bi‐ and tri‐cellular junctions could be clearly imaged and analyzed in terms of their frequency (no significant difference noted between bi‐ and tri‐cellular junctions) and duration (6 ± 0.5 minutes in IL‐1β‐stimulated tissues). Clear evidence for the formation of transcellular pores was also obtained (9.3 ± 2.1 % for all reactions). Collectively the high spatial and temporal resolution of our approach has led to novel qualitative and quantitative analysis of neutrophil TEM in vivo.

Funded by the Wellcome Trust (Ref: 081172/Z/06/Z to SN)
CORM‐3‐derived CO attenuates oxidative stress in vascular endothelial cells in SOD‐activity independent manner
Authors: Mizuguchi Shinjiro , Stephen Jancy , Katada Kazuhiro , Capretta Alfredo , Potter Richard F , Fraser Douglas F , Cepinskas Gediminas
Published: June 2021

Our previous studies indicate that CO derived from CO‐releasing molecule, CORM‐3, while suppressing PMN transendothelial migration at the same time amplifies oxidative stress in PMN, whereas oxidative stress in vascular endothelial cells (HUVEC) is effectively suppressed by CORM‐3. However, the mechanisms of such opposing effects of CO with respect to ROS production are unknown.

In this study we assessed the effects of CORM‐3‐derived CO on modulation of the antioxidant enzyme, superoxide dismutase (SOD), activity in an in vitro model of endotoxemia. To this end, HUVEC were stimulated with LPS (1μg/ml) for 1–4h in the presence or absence of CORM‐3 (100μM) and assessed for production of ROS, activation of NFκB and expression of adhesion molecule ICAM‐1. In parallel, SOD activity in cell lysates (cell‐associated SOD) and supernatants (soluble SOD) were also assessed.

The obtained results indicate that LPS‐induced increase in ROS production, activation of NFκB and ICAM‐1 expression were effectively reduced by CORM‐3. Interestingly, CORM‐3‐dependent suppression of ROS production was not associated with increased SOD activity. On the contrary, CORM‐derived CO attenuated SOD activity in both, cell lysates and supernatants, and in an assay employing purified SOD1 enzyme, indicating that CORM‐3‐derived CO negatively regulates SOD activity in cultured vascular endothelial cells (HSFO‐NA6171; IRF‐025‐09).
Autophagy is Important for the Mitigation of Ethanol‐Induced Hepatotoxicity and Steatosis
Authors: Liao Yong , Ding Wen‐Xing , Chen Xiaoyun , Ni Hong‐Min , Stolz Donna B , Yin Xiao‐Ming
Published: June 2021

Alcohol abuse is a major cause of liver injury. Whereas the pathology of alcoholic liver disease develops over a prolonged period the cellular defense mechanisms against the detrimental effects of alcohol are not well understood. We investigated whether macroautophagy, an evolutionarily conserved mechanism commonly activated in response to stress, could be a protective mechanism against ethanol toxicity. Using both an animal binge model and cell cultures, we found that ethanol treatment strongly activated macroautophagy. This induction relied on the metabolism of ethanol, ROS generation and inhibition of mTOR signaling. Suppression of macroautophagy with pharmacological agents or specific siRNAs led to a significant increase in hepatocyte apoptosis and liver injury, indicating that autophagy was a key defense mechanism in the mitigation of the toxic effects of alcohol. Further analysis demonstrated that macroautophagy protected hepatocytes by removing damaged mitochondria and accumulated lipid droplets. Thus enhancement of macroautophagy greatly reduced the hepatotoxicity and steatosis associated with acute ethanol exposure. This study established the physiological significance of macroautophagy in ethanol‐induced pathogenesis in the liver and the potential therapeutic values of modulating macroautophagy in alcoholic liver disease.
Role for β‐catenin in repair of biliary injury after exposure to DDC
Authors: Thompson Michael D , Singh Sucha , Monga Satdarshan
Published: June 2021

Diverse roles for the Wnt pathway in oval cell mediated liver injury repair exist. Atypical ductular proliferation (ADP) followed by the appearance of oval cells and liver fibrosis occurs in mouse liver following DDC‐induced biliary injury. We previously noted a blunted ADP in mouse liver lacking β‐catenin. To examine whether the presence of active‐β‐catenin would provide a growth or differentiation advantage in this model, liver‐specific β‐catenin over expressing mice (TG) with a mutation at ser45 were fed the DDC diet. No difference in appearance and number of A6‐positive (oval cell/biliary marker) cells was observed in the short‐term. However, we observed an increase in A6‐positive hepatocyte‐like cells in TG as early as 14 days and a dramatic difference at 150 days. We also observe an increase in PCNA positive hepatocyte‐like cells at 28 and 150 days. Coincident with this finding, we observe decreased serum bilirubin and alkaline phosphatase levels in TG mice indicating a resolution of biliary injury. It is conceivable that β‐catenin overexpression allows the liver to recover more quickly from injury. TG mice exposed to DDC for 4 weeks followed by 2 days of normal chow had less ADP and periportal inflammation compared to WT. These findings demonstrate the dexterity of β‐catenin overexpressing hepatocytes to repair biliary injury secondary to DDC exposure.
Haptic Simulation of the Feel of the Body Surface for Training in Palpatory Diagnosis
Authors: Howell John , Conatser Robert , Eland David , Williams Robert
Published: June 2021

Surface anatomy plays a special role in osteopathic medicine because palpatory diagnosis of musculoskeletal problems is prerequisite to manipulative treatment. As part of the physical exam, students learn to palpate the back for signs of dysfunction, which may be somatic or visceral in origin, and may be mediated by altered excitability of spinal neurons. The Virtual Haptic Back (VHB), a virtual reality simulation of the contours and textures of the human back, is used to help students learn palpatory diagnosis. Two haptic interfaces permit students to practice locating by touch regions within the paravertebral musculature of the simulated back having abnormal tissue texture (stiffness). The location of the abnormality varies randomly between trials, and the degree of compliance alteration also varies, allowing differences in task difficulty. In each trial students get immediate feedback as to the correctness of their diagnosis. Data from over 300 students demonstrate: 1) improvement in palpatory precision and speed over six, 15‐minute practice sessions, and 2) positive evaluations of the experience in terms of contributing to the development of student palpatory skills.
Identification and Initial Characterization of Histone Lysine Propionylation and Lysine Butyrylation Pathways
Authors: Zhao Yingming
Published: June 2021

Protein post‐translational modifications (PTMs) in histones play a crucial role in chromatin‐templated cellular processes. At least 10 types of histone PTMs were described in the past. However, it remains unknown if previously undescribed PTMs exist in histones. Using mass spectrometry, we recently identified lysine propionylation and lysine butyrylation as two novel PTMs in histones, which are structurally similar to lysine acetylation. We validated structures of these histone PTMs by three independent methods: (i) tandem mass spectrometry of synthetic peptides, (ii) co‐elution in HPLC, and (iii) Western blotting using sequence‐specific and pan antibodies. Using pan antibodies against the two PTMs, we showed that these two PTMs are abundantly present in core histones among several eukaryotic species we examined. We demonstrate that p300 and CBP, two acetyltransferases, can catalyze lysine butyrylation reactions, while HDAC3 is lysine debutyrylation enzyme for histone butyrylation. ChIP‐qPCR assay and ChIP‐on‐chip experiments indicate that histone lysine butyrylation is associated with transcriptional regulation and with various genomic locations in a similar fashion as lysine acetylation. Our results therefore provide the first snapshot of the new histone PTMs and suggest the higher complexity of the histone PTMs.
Biological Insights From Quantitative Analysis of Tyrosine Kinase Signaling Networks
Authors: White Forest M
Published: June 2021

To effectively monitor protein phosphorylation events governing signaling cascades, we have developed a mass spectrometry‐based methodology enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins at multiple time points under a variety of perturbations. We have recently applied this technique to identify key signaling nodes regulating EGFR, Insulin Receptor, and T Cell Receptor signaling network response to stimulation.

We have also performed an in‐depth characterization of the network‐wide effects of single‐point mutations on the oncogenic, constitutively active, EGFRvIII receptor tyrosine kinase. This study quantifies the adaptive capacity of cellular signaling networks and the pleiotropic effects of knocking out individual phosphorylation sites with the network. Computational analysis of the phosphorylation data relative to phenotypic data highlights the role of selected protein phosphorylation sites in regulating biological outcome. We also demonstrate the utility of a data‐driven computational model that is capable of fully describing glioblastoma cell growth based on just 10 phosphoproteins. Overall, we have now demonstrated that the combination of mass spectrometry‐based analysis of protein phosphorylation with phenotypic measurements and computational modeling yields novel insights into the regulation of cellular signaling on a network scale.
Molecular Mechanisms of Resistance to Imatinib or Sunitinib in KIT Mutants from Patients with Gastrointestinal Stromal Tumors
Authors: Wu Joe C
Published: June 2021

The activation of receptor tyrosine kinases (RTKs) is tightly regulated through a variety of mechanisms including autophosphorylation. Dysfunction of the regulatory mechanism frequently results in human diseases such as cancer. A large percentage of gastrointestinal stromal tumors (GISTs) are linked to aberrant activation of the receptor tyrosine kinase KIT, mainly driven by mutations in the juxtamembrane region of the protein. Tyrosine kinase inhibitors sunitinib and imatinib have been used for treatment of GISTs. While the drugs have demonstrated effective clinical benefit for GIST patients via inhibition of KIT, mutations emerge in the activation loop of KIT that render the protein insensitive to the drugs. Biochemical and structural studies that provide insight towards the mechanism of drug action, resistance and selectivity will be presented. In addition, the mechanism by which mutations result in kinase activation and the preferential binding mode of inhibitors to inactive vs. active forms of kinases will be discussed.
Dynamics of PKA Signaling
Authors: Taylor Susan S.
Published: June 2021

The protein kinases represent one of the largest mammalian gene families and regulate in diverse and critical ways most biological processes in all eukaryotic cells. cAMP‐dependent protein kinase (PKA) has served as a prototype for understanding the structure and function of this protein kinase superfamily. The PKA catalytic subunit is assembled into an active conformation by phosphorylation of its activation loop where this single phosphate has profound effects on global structure and function. All active protein kinases share a common architecture that is built upon two hydrophobic spines comprised of residues from both the small (N‐) and large (C‐) lobes. These spines are anchored to the hydrophobic F Helix, which traverses the large helical C‐lobe. The regulatory spine is typically assembled by phosphorylation of the activation loop while the catalytic spine is completed by the adenine ring of ATP. The active kinase then opens and closes as it navigates through the catalytic cycle. The PKA C‐subunits are regulated by dimeric regulatory (R) subunits, which form an inactive tetrameric complex in the absence of cAMP. Catalytic activity is unleashed by cAMP binding with high affinity to the R‐subunits. Structures of R‐subunit monomers bound to the C‐subunit demonstrated that the R‐subunit undergoes major conformational changes as it releases cAMP and binds to the C‐subunit. The cooperative binding of cAMP then releases the C‐subunit. Specificity in PKA signaling is achieved in part by the isoform diversity of the regulatory subunits and by targeting of the holoenzymes to specific sites by A Kinase Anchoring Proteins (AKAPs). The AKAP docking site is localized to the N‐terminal dimerization/docking (D/D) domain of the R‐subunits, a four helix bundle that binds with high affinity to an amphipathic helix in the AKAP. Structures of the D/D domains from RIIα and RIα bound to the same AKAP peptide from D‐AKAP2 reveal an isoform‐specific code for binding to AKAPs. Crystallization of tetrameric holoenzymes, coupled with small angle Xray an neutron scattering, demonstrates how full length holoenzymes are assembled and anchored to AKAPs and reveals for the first time the full allosteric potential of PKA signaling.

Support for this work was provided by NIH DK54441, GM19301, and GM34921.
The structure and function of ATP‐dependent chromatin remodeling complexes
Authors: Bartholomew Blaine , Dechassa Mekonnen Lemma , Hota Swetansu K , Prasad Punit , Sen Payel , Gosh Sujana , Hemeryck Christine S. , Pugh Frank , Pondugula Santhi , Kladde Michael P.
Published: June 2021

ATP‐dependent chromatin remodelers are key regulators of the epigenome and have important roles in transcription, replication, repair and recombination of DNA. These remodelers all have the basic ability to translocate along DNA in an ATP‐dependent manner. The remodelers use this activity in various ways to reorganize chromatin fibers through nucleosome mobilization, histone exchange, and/or nucleosome disassembly. The interactions with chromatin of three of the four major families of ATP‐dependent chromatin remodelers are examined to find some of their fundamental differences. The subunits and protein domains that bind nucleosomes have been mapped. The mechanism of chromatin remodeling has been studied using a variety of techniques to capture the intermediate states and to map changes in protein‐DNA and protein‐protein interactions. Deletion and point mutagenesis of protein domains have further delineated the functional role of particular domains in chromatin remodeling both in vitro and in vivo and has provided additional insights into the mechanism of chromatin remodeling. Three basic models for chromatin remodeling will be presented that account for some of the diversity among chromatin remodelers.
The dietary antioxidant resveratrol is a functional scavenger of peroxynitrite in vitro
Authors: Holthoff Joseph H , Burns Samuel T , Woodling Kellie A , Doerge Daniel R , Hinson Jack A , Mayeux Philip R
Published: June 2021

Resveratrol (Rsv) is a polyphenol found naturally in dietary products such as red wine and peanuts and suggested to have antioxidant properties. However, the mechanism(s) by which it may function as an antioxidant is unclear. Oxidants are a major contributor to cellular damage in numerous pathological states. We have previously shown that Rsv reduces the formation of NO‐derived reactive nitrogen species (RNS) in the kidney during sepsis. Thus, we propose that Rsv is able to protect against RNS‐mediated cellular damage by acting as an RNS scavenger. To test this, Rsv was reacted in vitro with varying concentrations of the RNS, peroxynitrite (ONOO−). LC/MS/MS revealed the formation of Rsv dimers, trimers, and nitrated Rsv products. Next, nitration of bovine serum albumin by ONOO− was used as a functional assay. Rsv blocked the formation of nitrotyrosine adducts on bovine serum albumin in a concentration‐dependent manner, indicating Rsv can act as a functional scavenger of ONOO−. Furthermore, Rsv was more potent at inhibiting nitration than the known RNS scavenger, N‐acetylcysteine. These data indicate that Rsv can act as a potent RNS scavenger and that this activity may explain its protective effects in the kidney during sepsis. Moreover, uncovering the RNS scavenging ability of Rsv contributes to the understanding of its other reported beneficial effects such as increasing longevity. Supported by NIH DK075991.
Identification of anti‐inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using mouse macrophages
Authors: Huang Nan , Hauck Cathy , Rizshsky Ludmila , Murphy Patricia A. , Nikolau Basil J. , Birt Diane F.
Published: June 2021

Ethanol extracts from eleven species and accessions of Hypericum were screened for their inhibitory properties on LPS‐induced prostaglandin E2 (PGE2) and nitric oxide (NO) in RAW264.7 mouse macrophages. All extracts inhibited PGE2 production by 19%–45% at 10 μg/ml compared to vehicle control while all but one extract also decreased NO production by 10%–35%. No cytotoxicity was noted on any extract at 30 μg/ml. Among all extracts, those from H. perforatum and H. gentianoides demonstrated stronger overall inhibitory potency. HPLC analysis of the extracts revealed that H. perforatum extract contained pseudohypericin, amentoflavone, quercetin and chlorogenic acid; previously identified as a ‘4 component system’; while H. gentianoides extract contained acylphloroglucinols including uliginosin A. H. perforatum and H. gentianoides extracts were fractionated using semi‐preparative HPLC and several fractions inhibited both LPS induced PGE2 and NO production dose‐dependently. HPLC analysis of the most active fractions indicated that ‘4 component system’ could be the major contributor to H. perforatum activity and suggested that acylphloroglucinols were potentially involved in H. gentianoides activity. This study demonstrated significant differences between two Hypericum species with regards to anti‐inflammatory compounds and potential mechanism of action, which will be confirmed by further study.

Grant Funding Source: P50 AT004155‐06 NCAAM/ODS from NIH
Arachidonate 5‐lipoxygenase(ALOX5) gene variants affect eicosanoid production and response to fish oil supplementation
Authors: Stephensen Charles Bolt , Armstrong P. , Legault J. , Schuster G. , Newman J. W. , Pedersen T. L. , Kelley D. S. , Hartiala J. , Allayee H.
Published: June 2021

Subjects with low intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) who are homozygous for 3 or 4 tandem repeats of an Sp1 binding site in the ALOX5 promoter have increased atherosclerosis relative to subjects homozygous for the common 5 repeat allele. To determine whether these alleles are functional, we recruited 116 adults with low (55) and high risk genotypes (33, 34, 44, 35, 45) and examined ALOX5 protein and metabolite levels in monocyte cultures. At baseline, ALOX5 protein did not vary by genotype whereas levels of arachidonic acid (AA)‐ and EPA‐derived ALOX5 metabolites 5‐hydroxyeicosatetraenoate (5‐HETE) and 5‐hydroxyeicosapentaenoate (5‐HEPE), respectively, were significantly higher in the 55 group compared to the other genotypes. No differences were seen for either AA‐derived leukotriene B4 (LTB4) or EPA‐derived LTB5. 98 subjects who were randomized within genotypes to treatment with 2g EPA/1g DHA per day or placebo (corn/soy oil) completed the study. Regardless of genotype, 5‐HETE and LTB4 levels did not change but LTB5 increased significantly in the EPA/DHA group after 6wk of treatment. 5‐HEPE levels also increased significantly in response to EPA/DHA with a greater effect in the 55 group. These results suggest that these ALOX5 alleles are functionally different with respect to AA/EPA metabolism, which may have important implications for heart disease risk.