bluuebunny/crossref_metadata_2025 huggingface dataset:
Young Healthy Humans Demonstrate Reduced Executive Function in Moderate Hypoxia
Authors: Buchholtz Zachary A. , Baker Sarah E. , Wiggins Chad C. , Trenerry Max R. , Clifton Haider R. , Holmes David R. , Joyner Michael J. , Curry Timothy B. , Uchida Koji
Published: April 2020

Background

Cognitive deficits resulting from mild hypoxia are difficult to quantify and reproduce consistently. The changes in executive function during acute hypoxia are still controversial. The purpose of present study was to assess changes in executive function during acute moderate hypoxia using a continuous performance test (Connors’ Continuous Performance test, CCPT‐II).

Methods

Nine young, healthy adults (7 women; 28 ± 1 years; BMI: 24.5 ± 3.0 kg·m−2) performed two sets of CCPT‐II during two different conditions: 1) 7 minutes of baseline (fraction of inspired oxygen (FiO2) = 0.21) and 2) 10 minutes of hypoxia (FiO2=0.118). The CCPT‐II is a test of executive cognitive function which requires subjects to strike a key when any letter except the letter X appeared on a computer monitor as quickly and accurately as possible. Reaction time, commission errors (response to X), and omission errors (failure to respond to a letter other than X) were recorded for each 140s of the CCPT‐II block. Arterial oxygen concentration (PaO2) was measured during the last 30sec of baseline and hypoxia from brachial artery catheter samples.

Results

PaO2 was reduced in hypoxia (baseline: 103.1 ± 1.5 mmHg vs. hypoxia: 43.2 ± 1.0 mmHg, P<0.01). During the trials of the CCPT‐II performed during hypoxia, reaction time was lower (i.e. faster) than during baseline (baseline: 451 ± 18 ms. vs. hypoxia: 437 ± 13 ms., P<0.01). Additionally, the number of commission errors was greater in hypoxia (baseline: 1.3 ± 0.6 errors·block−1 vs. hypoxia: 2.1 ± 0.8 errors·block−1, P<0.01). However, the number of omission errors was not different between the two conditions (baseline: 0.4 ± 0.2 errors·block−1 vs. hypoxia: 0.2 ± 0.1 errors·block−1, P=0.92).

Conclusions

The number of commission errors increased and the reaction time decreased during the CCPT‐II test performed in moderate hypoxia compared to baseline, demonstrating a decline in cognitive function during hypoxia. The faster and less accurate responses during hypoxia suggest a suppression of inhibitory pathways in the prefrontal or motor cortex that may contribute to the observed acute reduction in executive cognitive function.

Support or Funding Information

This project was funded by the Office of Naval Research (ONR), grant number N00014‐18‐D‐7001‐TO1.
Cell‐specific heterogeneity of Cx43‐positive gap junctions in the mammalian kidney
Authors: Xu Yan , Hu Junda , Horn John , Bachmann Sebastian
Published: April 2020

Objective

Connexin 43 (Cx43) is a gap junction‐forming protein which mediates the intercellular passage of molecules < 1000 Da. In the kidney, Cx43 was reported in glomerular vessels and mesangium. Own results prompted us to reevaluate the distribution of Cx43 and its phosphorylated form, phospho‐S368‐Cx43 (pCx43), within the different zones of the rat kidney and in cultured fibroblasts in more detail.

Methods

Cx43 and pCx43 were localized by immunofluorescence in rat kidney sections and cultured human fibroblasts. High‐resolution immunostaining of Cx43 was achieved by ultrastructural immunogold labelling on electron microscopy sections. Double‐labelings with anti‐aquaporin 1 (AQP1), aquaporin 2 (AQP2), zonula occludens‐1 (ZO‐1), and cyclooxygenase‐2 (COX‐2) were conducted to clarify Cx43 distribution in the rat renal medulla. Cultured human fibroblasts served to study intercellular transfer of fluorescent lucifer yellow.

Results

Cx43 was distributed in renal arterial and arteriolar endothelia, lymphatic vessel walls, peritubular capillaries, and glomerular compartments. In the interstitium, Cx43 was present as linear or punctate signals of the cell membrane of fibroblasts, in colocalization with pCx43. Cx43‐positive medullary fibroblasts co‐expressed COX‐2. No colocalization was detected between Cx43 and AQP1 or AQP2. Ultrastructural analysis showed Cx43 immunoreactivity at membrane contact zones between fibroblasts and in isolated cell processes of inner medullary interstitium. Cultured fibroblasts showed extensive, punctate Cx43 and pCx43 signals between adjacent cells. Signal was further distributed in non‐junctional areas of plasma membrane, suggesting Cx43 hemichannel functions. Lucifer yellow spread rapidly (10 min) between fibroblasts suggesting intercellular exchange via gap junctions, which was blocked by the gap junctional inhibitor, 18β‐glycyrrhetinic acid.

Conclusions

Cx43 in rat kidney shows prominent glomerular, vascular, and interstitial distribution, while epithelia, with exception of the podocytes, were negative. Focusing on interstitial fibroblasts, Cx43‐mediated information transfer was demonstrated. A constitutive nature of Cx43 phosphorylation was established in these cells. Our data suggest a cell‐specific heterogeneity of Cx43‐positive gap junctions mediating information exchange among renal cell types.
Simulated Stroke in a Mouse Neuronal Cell Culture Model
Authors: Meyer Luise J. , Eskaf Josephine , Salzman Michele M. , Li Zhu , Riess Matthias L.
Published: April 2020

INTRODUCTION

Despite many efforts, stroke remains a cause of debilitating illness and death worldwide. The fact that not only ischemia itself, but also the return of blood flow aggravates cellular dysfunction, illustrates a clinical barrier. Hence, catheter‐based interventions, the therapy of ischemic strokes, can add detrimental tissue damage, described as ischemia/reperfusion (I/R) injury. It is therefore crucial to develop potential pharmacological strategies that attenuate reperfusion injury and improve neurological function. Animal in‐vivo and in‐vitro models are vital to determine potential targets for pharmacological therapies. While in‐vivo models better mimic the complexity of the clinical context, in‐vitro studies allow to focus on the pathophysiology and can be performed on individual cell types. This study aimed to establish an in‐vitro stroke model by investigating the effect of simulated I/R injury on mouse brain isolated neurons.

METHODS

Neurons were plated and grown to confluency. To simulate stroke, cells were then randomized to either control/normoxic (21% O2, 5% CO2, 74% N2) or hypoxic conditions in a hypoxic chamber (0.01% O2, 5% CO2, 94.99% N2) in serum‐ and glucose‐free media. Durations of hypoxia for 2.5, 5 and 10h were compared. After 2h of reoxygenation in regular media and normoxic culture conditions following hypoxia/normoxia, samples were assayed for cell number/viability, cytotoxicity by lactate dehydrogenase (LDH) release and mitochondrial viability. Statistics: Mann‐Whitney Rank Sum Test and regression analysis; alpha=0.05.

RESULTS

Hypoxia significantly impaired cell number/viability after 5 and 10h, whereas 2.5h of hypoxia did not. A large decrease of cell number/viability was observed after 10h of hypoxia compared to 5h and 2.5h. The same observations were made for mitochondrial viability, apart from a slightly lower decrease after 5h hypoxia compared to the cell number/viability. Inverse relations were found for LDH release, except for an already significant increase in cytotoxicity after 2.5h of hypoxia.

CONCLUSIONS

Our data demonstrate that hypoxic injury in this model is time‐dependent. While 2.5h did not result in sufficient damage and 10h injured neurons too excessively, 5h hypoxia led to a moderate enough damage, still allowing for potential attenuation through protective strategies. Therefore, 5h hypoxia, followed by 2h reperfusion, appears to be a suitable duration for an in‐vitro mouse neuron stroke model to test new treatments.

Support or Funding Information

This work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.
The role of NFκB in pentagalloyl glucose inhibition of TNF‐α‐induced CXCL1 expression and induction of apoptosis‐related genes in racially different triple negative breast cancer cells.
Authors: Mendonca Patricia , Soliman Karam F.A.
Published: April 2020

Drug resistance is the leading cause of breast cancer‐related mortality in women, and triple negative breast cancer (TNBC) is the most aggressive subtype. CXCL1/GRO‐α has been identified as a pro‐inflammatory chemokine overexpressed in breast cancer, promoting angiogenesis and having an increased potential to metastasize. The current study investigated the effect of the naturally occurring polyphenolic compound pentagalloyl glucose (PGG) in TNF‐α‐induced CXCL1 expression in MDA‐MB‐231 (Caucasian) and MDA‐MB‐468 (African American) TNBC cells. In addition, PGG apoptotic effect was evaluated through the study of apoptosis‐related genes in both cell lines. Results showed that PGG inhibited TNF‐α‐stimulated CXCL1 expression in the transcription and protein level in Caucasian and African American TNBC cell lines. Through mRNA quantification analysis, data showed that PGG downregulated IKBKE (expressed in 60.4% of the breast cancer tumors) and MAPK1/ERK2 (expressed in tumor tissues with 5 to 10‐fold increase) expression in both cell lines. Moreover, PGG was more effective in inhibiting IKBKE expression in MDA‐MB‐231 compared to MDA‐MB‐468 (9 and 2‐fold inhibition, respectively). Also, PGG reduced MAPK1 gene expression in 3‐fold in Caucasian and 6‐fold in the African American cells. Gene transcription studies showed that PGG induced expression of the pro‐apoptotic genes BNIP3, BNIP3L, GADD45A, TP53BP2, and RIPK2, which are critical initiators of the intrinsic and extrinsic apoptotic pathways. Although PGG increased the expression of apoptosis‐related genes in both cell lines, there was a significantly higher expression in MDA‐MB‐231 cells (5 to 14.7‐fold) compared to MDA‐MB‐468 cells (2 to 5.8‐fold). Furthermore, BAK1 gene expression was only induced in African American cells showing a 35.7‐fold increase after PGG treatment. The obtained data show that PGG inhibited the expression of CXCL1 pro‐inflammatory chemokine and upregulated the expression of specific pro‐apoptotic genes. It was concluded that PGG might have great potential in target therapy for TNBC.

Support or Funding Information

This work was supported by a grant from the NIH/NIMHD grant U54 007582
A Mechanistic Omics‐based Evaluation of Mitochondria‐targeting‐H2S‐donor (AP39) Against Doxorubicin Cardiotoxicity
Authors: Veeranki Sudhakar , Scott David A.
Published: April 2020

Use of the highly effective broad‐spectrum anti‐cancer chemotherapeutic agent, doxorubicin (Dox), induces Dox‐induced cardiotoxicity (DIC) which leads to mortality and morbidity in pediatric cancer survivors. Dox treatment alters cardiac metabolism and mitochondrial function, however, underlying mechanisms and prevention strategies have not yet been discovered, which limits Dox’s broad clinical utilization. Our data demonstrated that Dox, along with mitochondrial toxicity, depleted both mitochondrial hydrogen sulfide (H2S) and mitochondrial localized H2S producing enzyme, 3‐mercaptopyruvate sulfurtransferase (MPST). As H2S is a cardioprotective signaling molecule, we tested the ability of mitochondria‐targeting‐H2S‐donor, AP39, to protect from the DIC. AP39 treatment was highly effective in mitigating the DIC in mouse models. Further, the combined use of RNAseq, targeted metabolomics and 13C‐labeled‐glucose‐isotope tracing approaches in a cardiomyoblast cell line revealed that AP39 conferred protection against dox‐toxicity by reversing key transcriptional and metabolic features. Moreover, there were improvements in mitochondrial antioxidant status, H2S presence, and quality control. Thus, AP39 is instrumental in protecting mitochondria and promoting stress‐resistant metabolism against DIC. In summary, the integrated omics‐based approach revealed that AP39 confers potent cardioprotection against DIC.

Support or Funding Information

The study was supported by the funds from the American heart association award# 17SDG33670578 to Dr. Veeranki. Dr. Veeranki is also supported by NIH grant HL131782 to Douglas Andres (D.A.) and Jonathan Satin (J.S.) (University of Kentucky). We thank Bryana Levitan (University of Kentucky) for the help with Echocardiography. We thank D.A. and J.S. for sharing some reagents. We thank Markey cancer center RM SRF (supported by NCI Cancer Center Support Grant (P30 CA177558)) for the help with OCR assays. The Vevo 3100 was supported by the Saha Cardiovascular Research Center.
TRPC6 deficiency or feeding a high fat diet impairs ventilatory responses to hypercapnia
Authors: Moak Sydney P. , Wang Zhen , da Silva Alexandre A. , Dai Xuemei , Hall John E. , do Carmo Jussara M.
Published: April 2020

Transient receptor potential cation channel C (TRPC) is essential for hypoxic pulmonary vasoconstriction response and alveolar gas exchange. We previously showed that whole body TRCP6 deficient (TRPC6 KO) mice are obese and resistant to the anorexic effects of leptin, a hormone that modulates ventilatory control. In the present study we investigated ventilatory responses to hypercapnia (7% CO2) in male and female 22‐week‐old TRPC6 KO and B6/129 control mice fed normal or high fat diet (HFD) since weaning (n=5 per group/sex/diet). Pulmonary ventilation (VE), tidal volume (VT) and respiratory frequency (FR) were measured using the plethysmography method. TRPC6 KO mice were heavier than control mice when fed normal diet (46.2±1.9 vs. 36.6±2.2 g in males and 34.0±1.4 vs. 26.9±1.3 g in females). However, when fed a HFD, male and female TRPC6 KO and B6/129 mice had similar body weight (43.6±0.4 vs. 47.5±1.4 g and 40.1±0.6 vs. 40.7±2.5 g, respectively). Compared to B6/129 controls male and female TRPC6 KO mice fed normal diet had similar VE (1817± 233 vs. 2139±135 and 2146± 404 vs. 2943±421 ml/min/kg) and VT (10.9±0.6 vs. 9.9±0.6 and 17.0±3.4 vs. 14.6±0.8 ml/kg), but lower FR (130±9 vs. 217±14 and 127±5 vs. 201±26 breaths/min) at room air. During hypercapnia, only male TRPC6 KO mice exhibited lower VE (3631± 493 vs. 6003±698 ml/min/kg), which was due to lower FR (235 ±16 vs. 305±19 breaths/min) and VT (15.3±1.0 vs. 19.5±1.4 ml/kg) responses to 7% CO2. Although female TRPC6 KO mice also showed lower FR to hypercapnia compared to B6/129 controls (242±6 vs. 313±22 breaths/min) they exhibited higher VT (28.4±3.0 vs. 25.8±2.0 ml/kg), thus resulting in similarly increased VE (6914±847 vs. 8119±956 ml/min/kg). Male and female B6/129 mice fed a HFD had significantly reduced VE responses to 7% CO2 (4020± 332 vs. 6003±698 and 5140± 194 vs. 8119±956 ml/min/kg, respectively) that were mainly caused by reduced VT (14.4±1.3 vs. 19.5±1.4 and 18.8±0.6 vs. 25.8±2.0 ml/kg). No further reduction in in ventilatory responses to hypercapnia were observed in male or female TPRC6 KO mice fed a HFD. These results suggest that TRPC6 plays an important role in ventilatory responses to hypercapnia, albeit more profound in males than females, and that HFD significantly reduces ventilatory responses to hypercapnia in both male and female control mice, but not in TRPC6 KO mice. The mechanisms involved in the reduced ventilatory responses are still unclear but may involve reduced sensitivity to leptin evoked by HFD or TRPC6 deficiency.

Support or Funding Information

NIDDK 1RO1121411, NHLBI‐PO1HL51971, NIGMS P20GM104357 and U54GM115428
Adaptations to cursoriality and digit reduction in the forelimb of the African wild dog (Lycaon pictus)
Authors: Koshy Rahul , Alwiel Ryan , Adrian Brent , Grossman Aryeh , Smith Heather F.
Published: April 2020

The African wild dog (Lycaon pictus), a rare canid native to southern and eastern Africa, lives and hunts in complex social groups. It is currently classified as Endangered on the IUCN Red List of Threatened Species, with approximately 1400 mature individuals present in the wild. L. pictus is distinct among canids in lacking a digit I or “dew claw” and in its use of exhaustive predation to capture its prey instead of speed, strength, or stealth. These behavioral and morphological traits suggest a potentially unique set of adaptations. This study is the first detailed anatomical description and functional analysis of the forelimbs of L. pictus.

The forelimb myology of L. pictus is generally similar to other canids; however, it shows some notable exceptions. While a complete digit I is absent in L. pictus, a vestigial first metacarpal is present, resulting in changes to insertions of mm. extensor digiti I et II, abductor (et opponens) digiti I, and flexor digiti I brevis. M. triceps brachii caput laterale had a larger origin in L. pictus compared to other carnivorans, and m. triceps brachii caput longum had an additional accessory head. Electromyographic (EMG) studies have shown this muscle is active during the stance phase of trotting and galloping and is important for storing elastic energy during locomotion. Mm. anconeus, brachialis, and flexor carpi ulnaris caput ulnare all had more extensive origins in L. pictus than other canids. In domestic dogs, these muscles have a relatively high proportion of Type 1 (slow‐twitch) fibers and are important for posture and elbow stability. Statistical analyses of the quantitative muscle data reveal similar proportions of antebrachial muscle groups to other large‐bodied carnivorans. However, L. pictus has relatively smaller wrist rotators than any other included taxon, suggesting a need for stability over rotatory movement in the carpus of L. pictus.

We interpret these differences in size and attachments of muscles in L. pictus compared to other canids as adaptations for long distance running in this highly cursorial species, likely important for exhaustive predation. Absence of a digit I in L pictus, typically used to reduce torque during quick turns and for lightly gripping onto objects, may be related to a reduced need for gripping and quick agile movements in its cursorial lifestyle.

Support or Funding Information

This project was funded by Midwestern University, including a Kenneth A. Suarez Summer Research Fellowship to RK.
Role of TRPV4 mechanosensing in foreign body response and giant cell formation
Authors: Rahaman Shaik O. , Arya Rakesh K. , Goswami Rishov
Published: April 2020

Implantable biomaterials and medical devices are used in millions of procedures each year worldwide. However, the implantation of these devices leads to the development of a foreign body response (FBR), a chronic inflammatory condition that can ultimately lead to implant failure, which may cause harm to or death of the patient. Despite decades of research, the mechanisms underlying the FBR remain poorly understood, as neither the materials from which these implants are made nor their chemical properties can explain triggering of the FBR. Hallmarks of the FBR include activation of macrophages at the tissue‐implant interface, formation of destructive foreign body giant cells (FBGCs), and development of fibrous tissue that encapsulates the implant. Here, we report that genetic ablation of TRPV4, an ion channel in the transient receptor potential vanilloid family, and which is a mechanosensor, is possibly the mediator of FBR. Specifically, we found that: 1) Trpv4 deletion in mice prevented macrophage accumulation, FBGC formation, and collagen accumulation in a subcutaneous implantation model; 2) the severity of the in vivo macrophage accumulation at the tissue‐implant interface was dependent on the stiffness of the implant; 3) genetic ablation of TRPV4 blocked macrophage adhesion and spreading on stiff matrix in bone marrow derived macrophages; and 4) genetic deficiency of TRPV4 blocked cytokine‐induced FBGC formation, which was restored by lentivirus‐mediated TRPV4 reintroduction. Furthermore, we found that: 5) TRPV4 activity (calcium influx) was augmented in response to a biomechanical stimulus; and 6) loss of TRPV4 activity reduced internal stiffness in macrophages as determined by Atomic Force Microscopy analysis. Taken together, these results suggest an important, previously unknown role for TRPV4 in FBR and giant cell formation.

Support or Funding Information

Funding: This work was supported by NIH (1R01EB024556‐01) and NSF (CMMI‐1662776) grants to Shaik O. Rahaman.
A Tool to Predict Affinity and Specificity of GAG Binding to Proteins
Authors: Desai Umesh R.
Published: April 2020

Glycosaminoglycans (GAGs) are unusual biopolymers that interact with a large number of proteins that reside intracellularly, extracellularly or on cell membranes. These include growth factors, chemokines/cytokines, serine proteases, serpins, metalloproteases, histone acyltransferases, integrins and other cell membrane receptors. Antithrombin is the only serpin believed to bind GAGs with high specificity. However, our computational algorithm is beginning to challenge this prejudicial assumption. In fact, our computational tool shows that GAG display selectivity from both – chemical as well as biological – perspectives. Alternatively, distinct group of GAG sequences selectively recognize a target protein from the horde of sequences (chemical specificity) and distinct binding sites on a protein uniquely recognize a group of GAG sequences (biological specificity). This understanding has come about from the computational tool we have developed to study GAG – protein interactions. The use of this novel combinatorial virtual library screening (CVLS) tool to predict affinity and specificity of GAG sequences for target proteins will be presented. In principle, the tool could be applied to any protein – GAG system of interest to identify the precise GAG sequence that offers high selectivity and high affinity.

Support or Funding Information

U01 CA241951; K12 HL141954; R25 HL128639; R01 HL090586
Genomic Profiling and Physiological Approaches to Understand Aquaporins and their Role in ROS Signalling within Skeletal Muscle
Authors: Staunton Caroline Amy , Vasilaki Aphrodite , McArdle Anne , Jackson Malcolm , Barrett-Jolley Richard
Published: April 2020

Aquaporin (AQP) channels are a family of integral membrane proteins shown to aid the movement of hydrogen peroxide (H202) across membranes
[1]. Their role in skeletal muscle is of key importance since intracellular generation of H202 and other reactive oxygen species (ROS) occurs in response to muscle contractions. These ROS initiate signalling processes and activate of a number of transcription factors
[2,
3]. During ageing, excessive levels of ROS have pathological effects, inducing muscle degradation, loss of neuromuscular junction integrity and loss of neuromuscular transmission.

Our studies
[4], as well as those of others, has identified the sources, sinks and functions of H202 in muscle but exactly how H202 is transported from its site of origin within muscle has never been examined. We hypothesize that (AQPs) are the primary means of regulating H202 movement through skeletal muscle membranes. This study has therefore examined expression of AQP isoforms in muscle and how they may change with age.

RNA transcripts from Tibialis Anterior (TA) muscle from control adult (6–8 months), and old (26month) mice, and from mice following peroneal nerve crush, were examined by RNAseq (150bp‐read depth >280 M clusters per lane, n=5). Individual muscle myotubes were also treated pharmacologically with aquaporin inhibitors and transfected with AAV6‐HyPer2 to determine the role of different AQP isoforms on intracellular H202 content and H20 permeability was determined using the swell assay
[5,
6].

RNAseq data identified 8 AQP mRNA transcripts present in TA muscle. A decrease in AQP4 and AQP1 and an increase in AQP3 contents were found following nerve crush (n=5, P<0.01) and this was confirmed by immunological techniques. The localisation and roles of these aquaporins is currently being investigated to determine whether they have protective functions or contribute to the age‐related decline skeletal muscle.

An exponential increase in intracellular H2O2 levels was recorded when AAV6‐Hyper2 transfected muscle fibres were exposed to 10μM H2O2. Pre‐treatment of muscle cells with AQP blockers HgCl2 (30μM), TEA (3mM) or Bumetanide (5μM) substantially decreased the rate of increase in intracellular H202 content. These data demonstrate the ability of aquaporins to regulate skeletal muscle H2O2 permeability. Challenging cells with an 80mOsm solution in conjunction with pharmacological AQP1 inhibitors also demonstrated a significant reduction in the rate of increase in cell volume.

In summary, these data demonstrate a potential important role for AQPs in muscle. Further work using AAV‐shRNA to knockdown specific AQPs is being utilised to identify the major AQPs that function in skeletal muscle.

Support or Funding Information

This study was supported by the MRC and NIA (AG051442).
Noteworthy Idiosyncrasies of 1α,25‐Dihydroxyvitamin D3 Kinetics for Extrapolation from Mouse to Man
Authors: Pang K. Sandy , Noh Keumhan , Yang Qi Joy , Sekhon Lavtej , Quach Holly P. , chow Edwin C.Y.
Published: April 2020

Calcitriol or 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3] is the active ligand of the vitamin D receptor (VDR) that plays a vital role in health and disease. Vitamin D is converted to the relatively inactive metabolite, 25‐hydroxyvitamin D3 [25(OH)D3] by CYP27A1 and CYP2R1 in the liver, then to 1,25(OH)2D3 by a specific, mitochondrial enzyme, CYP27B1 (1α‐hydroxylase) that is present primarily in the kidney. Degradation of both metabolites is mostly carried out by the more ubiquitous mitochondrial enzyme, CYP24A1. Despite that calcitriol inhibits its formation and degradation, allometric scaling revealed strong interspecies (mouse, rat, dog and human) correlations of the net calcitriol clearance (CL from dose/AUC∞), production rate (PR), and basal, plasma calcitriol concentration (CBL) with body weight (BW). A closer scrutiny of the collated literature data revealed that the basal plasma concentration of calcitriol was not taken into consideration in the estimation of AUC∞ (with subtraction of CBL) and CL after exogenous calcitriol dosing in both animals and humans. This led to an overestimation of AUC∞ and underestimation of CL. After correction, the allometric equations [plots of log(CL) or log(PR) versus log(body weight) or log(BW)] were: CL=0.654xBW0.807 and PR=36.6xBW0.718. The CL of calcitriol was further influenced by diseased states, and was found to be reduced in chronic kidney disease but increased in cancer when compared to healthy humans. These changes, however, were not discerned with allometric scaling since all clinical data appeared as a cluster of closely similar values on the allometric plot. PBPK‐PD (physiologically‐based pharmacokinetic‐pharmacodynamic) modeling (Ramakrishnan et al 2016), however, confirmed the dynamic interactions between calcitriol and Cyp27b1/Cyp24a1, showing the increase in CL as well as decrease in PR in a dose‐dependent fashion for the intravenous, murine data of Quach et al (2015). The PBPK model was successful in predicting data from the escalating oral and intravenous doses of calcitriol given to cancer patients upon scale‐up.
A Novel Role for the ZNF16 Protein in rRNA Transcription
Authors: George Chelsea L. , Diaz-Martinez Laura A.
Published: April 2020

The zinc finger protein 16 (ZNF16) has been reported to be involved in megakaryocytic and erythroid differentiation of pluripotent K562 leukemia cells. Analysis of the role of ZNF16 in these cells identified functions in cell cycle regulation and c‐KIT transcription. However, ZNF16 has ubiquitous expression in multiple tissues, thus suggesting that it might have additional functions. We explored potential roles for ZNF16 using a battery of assays to test for localization, roles in cell cycle, apoptosis and DNA repair, among others. Endogenous ZNF16 localizes to the nucleus and is enriched in the nucleolus. Interestingly, exogenous ZNF16 showed two patterns of localization depending on the tag used. ZNF16 tagged with a single EGFP (Enhanced Green Fluorescent Protein) showed nuclear localization while ZNF16 tagged with three EGFPs was mainly localized to the nucleoli and other subnuclear structures. Based on its nucleolar localization, we explored potential roles for ZNF16 in nucleolar structure, response to nucleolar stress, rRNA transcription, DNA damage, and DNA repair. Preliminary results hint at a role for ZNF16 in rRNA transcription. These results reveal a novel role for ZNF16 in basic cellular processes, consistent with its ubiquitous expression in human tissues. Further characterization at the cellular and molecular level will offer insight on the function of this poorly characterized gene.

Support or Funding Information

UTEP‐URI program; BD Biosciences Cancer Research Grant; Murdock Foundation and Gonzaga Start‐up funds.
TNFα Exposure Decreases Mitochondrial O2 Consumption in Motor Neurons
Authors: Heino Amanda Denise , Yap Jane Denise , Brown Alyssa Denise , Rana Sabhya Denise , Fogarty Matthew J. , Mantilla Carlos B. , Sieck Gary C.
Published: April 2020

Neuroinflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial O2 consumption. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. The cells were then exposed to TNFα (20 ng/ml) for 24 h. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a Nikon A1R confocal microscope (60x oil immersion 1.4 NA). After treatment with TNFα, mitochondria appeared fragmented but mitochondrial volume density increased by ~50%, as compared to control untreated cells. A Agilent Seahorse XF Analyzer was used to determine the O2 consumption of treated and untreated cells during a stress test that determines basal respiration, ATP production and maximum respiration. O2 consumption was normalized to mitochondrial volume density in both treated and untreated groups. After TNFα exposure, basal respiration, ATP production and maximum respiration decreased per mitochondrion. However, overall O2 consumption of the cells was unchanged after TNFα exposure due to an offsetting increase in mitochondrial volume density. Using MitoSox, we also measured reactive oxygen species (ROS) formation in treated and untreated NSC‐34 cells. ROS is a byproduct of mitochondrial O2 consumption, and can cause cellular damage. After TNFα exposure, ROS formation increased compared to untreated cells. We conclude that mitochondrial biogenesis and an increase in mitochondrial volume density represent a homeostatic mechanism to reduce O2 consumption the per mitochondrion, thereby damping ROS formation while maintaining overall cellular O2 consumption and ATP production.

Support or Funding Information

Supported by NIH grant AG44615 and HL105355.
Defining how viruses manipulate lipid phosphoinositides through activation of PI4P kinases to mediate viral replication
Authors: Burke John E. , McPhail Jacob A. , Rathinaswamy Manoj , Jenkins Meredith L.
Published: April 2020

Phosphoinositide kinases are central regulators of myriad cellular processes, and their misregulation is causative of multiple human diseases. We are currently focusing on the structural basis for the regulation of phosphoinositide kinases, specifically the Golgi localised phosphatidylinositol 4‐kinase beta (PI4KB). We have identified a previously uncharacterised protein binding partner of PI4KB, the GTPase regulatory protein c10orf76
1, and revealed novel insight into an unexpected PI4K positive feedback loop. Using a combined hydrogen deuterium exchange mass spectrometry (HDX‐MS) and structural biology approach we have defined the molecular basis of regulation by the protein c10orf76, revealing novel insight into the role of these PI4KB in viral replication. This has revealed key insight into unexpected feedback loops with Ras superfamily GTPases. Overall, this approach has provided a novel toolbox of engineered mutations to tease out the molecular basis of regulation of phosphoinositide kinases in both homeostasis and disease, and has revealed novel opportunities for the development of small molecule therapeutics for multiple pathogenic viral infections.

Support or Funding Information

Research was supported by the Canadian Institute of Health Research (CRN‐142393), and salary award for the CIHR new investigator and MSFHR scholar programs.
Endothelial‐derived microparticles activates TLR4/JAK3/STAT3 pathway to induce acute lung injury
Authors: Ou Zhijun , Yuan Hao-Xiang , Li Yu-Quan , Wang Yan-Sheng , Li Yan , Ma Jian , Xu Jun , Ou Jing-song
Published: April 2020

We previously reported that endothelial‐derived microparticles (EMP) could induce acute lung injury (ALI) and the mechanism is still not fully understood. Toll‐like receptor 4(TLR4) cascade has been considered to be involved in innate immune inflammation. As its downstream, janus kinases/signal transducers and activators of transcription (JAK/STAT), acts as a key signal pathway for immune responses. However, it remains unclear whether systemic circulating EMP could trigger TLR4‐pomoted JAK/STAT signaling contributing to ALI. EMP were isolated from human umbilical vein endothelial cells (HUVECs) stimulated with plasminogen activated inhibitor‐1. HUVECs were treated with/without different concentration of EMP. EMP induced inflammatory cytokines in cultured HUVECs. EMP could activated TLR4 and the downstream JAK3/STAT3 pathway. Silencing TLR4 inhibited the EMP‐induced JAK3/STAT3 pathway. EMP‐induced inflammatory response in plasma and bronchoalveolar lavage fluid, inflammatory cells infiltration. edema and JAK3/STAT3 pathway in the lung tissue, and ALI in C57BL6 mice. However, These effect induced by EMP were depressed in TLR4−/− mice. Our data demonstrated that EMP induce ALI by activating TLR4/JAK3/STAT3 pathway to promote inflammatory response. Our findings present the novel mechanisms by which EMP induce ALI.

Support or Funding Information

This research was financially supported by the National Natural Science Foundation of China (Grants 81670392, 81600382, 81770241, 81830013, 81970363 and Distinguished Young Scholar Grant 81325001), 973 project (2009CB522104) and International Cooperation project (2015DFA31070) from the Ministry of Science and Technology of China, Guangdong Natural Science Fund Committee (Grant 2015A030312009), the Changjiang Scholars Program from the Ministry of Education of China, the Sun Yat‐sen University Clinical Research 5010 Program.
Large Hypertrophy but Unmodified Specific Tension of Single Fibers of Body Builders
Authors: Monti Elena , Toniolo Luana , Marcucci Lorenzo , Martellato Ivan , Bondì Michela , Franchi Martino , Narici Marco , Reggiani Carlo
Published: April 2020

Introduction

Muscle hypertrophy is the main outcome of training for body building. Previous studies showed a discrepancy between the functional (force/tension) and structural (cross‐sectional area, CSA) proprieties of leg extensor muscles measured in vivo and the corresponding parameters measured in single muscle fibers
[1]
[2]. In particular, a puzzling finding from these studies was that of a lower specific tension of skinned fibers of body builders (BB) compared to a control (C) population.

In this study we aimed to investigate the possible reasons behind the alleged lower specific tension in single fibers of a strictly diet‐and‐training‐controlled BB population.

Methods

Five male body builders (26±4.7 yr; 87.2±9.7 kg; 178.4±7.5 cm) with a training history of at least 5 years were recruited for this study. Five age‐matched recreationally active males (24.6±3.6 yr; 84.4±14.9 kg; 186.4±4.4 cm) were recruited as controls. After ethical approval, biopsies from the vastus lateralis (VL) muscle were collected together with in‐vivo quadriceps maximum isometric voluntary contraction (MVC) and architecture and CSA. Data analysis included (i) single skinned fiber mechanics (n=200 fibers; maximum force, CSA, specific tension and calcium diffusion), (ii) single fiber myosin quantification (n=100 fibers), (iii) slow and fast fiber CSA via histological section analysis. In addition, swelling ratio (skinned fibers CSA/histological CSA) was calculated for each fiber type and for each subject. A general linear mixed model was used to assess statistically significant differences.

Results

Quadriceps MVC and CSA were 64% and 33% higher in BB than C (p<0.05), VL pennation angle (PA) and thickness (MT) were 16% and 15% higher in BB than C (p<0.05). Quadriceps specific force was 25% higher in BB than C (n.s.). Single skinned type 2 CSA and force were 48% and 28% higher in BB than C (p<0.05), while specific tension was 19% lower (p<0.05). Type 1 fibers specific tension was 32% higher in BB than C (p<0.05). Myosin content and calcium diffusion were similar among populations. CSA determined in histological sections was 10% and 27% higher in BB than C (p<0.05) for type1 and 2 fibers, respectively. When type 1 and type 2 fiber tensions were corrected for individual swelling ratio, the difference between BB and C disappeared.

Conclusions

Although at first sight these findings seem to confirm that in type 2 skinned fibers of BB specific tension is lower than in C, once accounting for fiber swelling, this difference disappears. This is further confirmed by the lack of difference in myosin concentration and calcium diffusion kinetics.

In contrast, the results point to the existence of a differential swelling response to the skinning process by BB and C fibers, possibly due to adaptations of the fiber cytoskeleton proteins or specific permeability of the membrane of slow and fast fibers.
SPARC, a Muscle‐Secreted Protein, Improves Glucose Tolerance via AMP‐activated Protein Kinase in Skeletal Muscle
Authors: Aoi Wataru , Sakuma Kunihiro , Krook Anna
Published: April 2020

Secreted protein acidic and rich in cysteine (SPARC) is an exercise‐responsive myokine. Although SPARC has been implicated in nutrient metabolism, the effect of this myokine on muscle metabolism is unclear. Here we investigated the effect of SPARC on glucose metabolism in skeletal muscle.

SPARC‐knockout mice showed impaired systemic glucose metabolism. Chronic SPARC treatment improved systemic glucose tolerance and AMP‐activated protein kinase (AMPK) signaling in skeletal muscle of high‐fat diet‐induced obese mice. A protein interaction assay showed that SPARC strongly interacted with voltage dependent calcium channel. In cell culture experiments, SPARC increased glucose metabolism concomitant with activation of AMPK, and this effect was suppressed by inhibiting the calcium‐dependent signaling.

Our findings suggest that SPARC treatment mimics the effects of exercise on glucose tolerance by enhancing AMPK‐dependent glucose uptake in skeletal muscle.

Support or Funding Information

This study was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI: Grant‐in‐Aid for Scientific Research (B) (grant numbers 25282199 and 17H02176).
MEF2C Mediates Chronic Pain in Rodent Neuropathy Models
Authors: Serafini Randal Alexander , Sakloth Farhana , Pryce Kerri , Bertherat Feodora , Akbarian Schahram , Zachariou Venetia
Published: April 2020

Peripheral nerve injury triggers a number of maladaptive changes in both peripheral and central sites, leading to debilitating hypersensitivity, as well as cognitive, motivational, and affective deficits. Understanding the epigenetic and transcriptional events mediating pathological pain and synaptic reorganization will provide essential information for novel approaches towards the management of neuropathic pain. Our lab has previously demonstrated that prolonged peripheral nerve injury leads to an upregulation of histone deacetylase 5 (HDAC5) in the mouse Nucleus Accumbens (NAc), and revealed a dynamic role of HDAC5 in the antiallodynic efficacy of monoamine‐targeting antidepressants. HDAC5 negatively regulates the expression of Myocyte Enhancer Factor 2C (MEF2C), a transcription factor critical for synaptic plasticity in adulthood. Aberrant MEF2C expression contributes to clinical neuropsychiatric abnormalities such as autism and schizophrenia. By utilizing qPCR and RNAscope in situ hybridization, we found that long‐term peripheral nerve injury dynamically affects MEF2C expression in distinct cellular populations of chronic pain circuits. We also used adeno‐associated vectors to overexpress MEF2C in the NAc of adult mice in order to determine the impact of this transcription factor in the trajectory of sensory hypersensitivity associated with peripheral nerve injury. We found that while MEF2C overexpression did not affect the induction of sensory hypersensitivity, it did disrupt the maintenance of sensory hypersensitivity symptoms. Specifically, MEF2C over‐expressors recover from mechanical allodynia and show restored motivational drive. In conclusion, our findings suggest that manipulation of the plasticity‐mediating transcription factor, MEF2C, in the brain reward system holds promise for interrupting the chronification of pain, as well as its affective components. We are further investigating the mechanisms underlying neuropathy‐related changes in MEF2C expression and/or activity via phosphorylation‐specific protein analysis, in vivo DNA methyltransferase identification (DamID), and gene silencing tools for downstream molecular targets.

Support or Funding Information

NS086444 & NS111351 NINDS,MSTP T32 NIH T32 GM007280,Irene & Eric Simon Brain Research Foundation Fellowship
The Influence of Selenium on Arsenic Hepatobiliary Transport
Authors: Zhou Janet R. , Kaur Gurnit , Arutyunov Denis , Leslie Elaine M.
Published: April 2020

Over 200 million people worldwide are exposed to the proven human carcinogen arsenic at levels exceeding the World Health Organization guideline of 10 μg/L. In animal models arsenic and selenium are mutually protective via the formation and biliary excretion of the seleno‐bis (S‐glutathionyl) arsinium ion [SeAs(GS)2]−, which allows for the fecal elimination of both compounds. Consistent with this, selenium deficiency in humans living in arsenic endemic regions is associated with an increased risk of arsenic‐induced disease. These observations have led to the initiation of human selenium supplementation trials. Despite these ongoing trials in arsenic endemic regions, the influence of selenium on human hepatic handling of arsenic is not adequately understood. Furthermore, supplementation trials have utilized different chemical forms of selenium with unknown influence on efficacy. In the liver, multidrug resistance protein 2 (MRP2/ABCC2) transports arsenic metabolites, including [SeAs(GS)2]− into bile, and the related MRP4 (ABCC4) transports other arsenic metabolites into sinusoids. We hypothesized that selenium will increase the biliary excretion of arsenic from HepaRG cells, an immortalized cell line used as a surrogate for primary human hepatocytes. To test this hypothesis, we studied the influence of selenite (SeIV), selenide (SeII), methylselenocysteine (MSC) and selenomethionine (SeMet) on arsenic efflux from HepaRG cells. The expression of genes involved in hepatic arsenic methylation (As3MT) and export (ABCC2 and ABCC4) were assessed. Crude membrane preparations subjected to immunoblots were used to evaluate the presence of MRP2 and MRP4 proteins. The influence of arsenic on the levels of these proteins was also assessed similarly. Fluorescence microscopy after treatment with 5(6)‐carboxy,2’,7’‐dichlorofluorescein (CDF) diacetate was performed to visualize the canalicular networks and assess MRP2 function. Transport across sinusoidal and canalicular membranes was measured after treatment of HepaRG cells with 1 μM 73AsIII ± selenium (in the forms SeIV, SeII, MSC or SeMet) using B‐CLEAR® technology. Biliary excretion indices (BEIs) were calculated to quantify the extent of arsenic export into bile. ABCC2, ABCC4, and As3MT are expressed by HepaRG Cells. MRP2 and MRP4 proteins were detected, and their expression was increased by the presence of AsIII. CDF accumulation in canalicular networks suggested that MRP2 was functional. At a 5 minute time point, the BEI of 73AsIII alone was 17%. The addition of SeII increased biliary excretion of 73AsIII to 30%. Biliary excretion of 73AsIII was lost in the presence of SeIV, SeMet and MSC. Arsenic underwent biliary excretion in HepaRG cells and this was stimulated by SeII, and inhibited by SeIV, SeMet and MSC. These data have implications for the form of selenium utilized for selenium supplementation trials in arsenic endemic regions.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)
Back to the Drawing Paper : Active Learning Approaches That Are Not All Boring
Authors: Severson Arlen R.
Published: April 2020

The most traditional approach for delivering gross anatomical information today is the traditional lecture and/or laboratory cadaver dissection or prosection. The anatomy laboratory provides the most active learning approach to learning body structure and function. Some anatomical concepts, however, cannot be visualized in a dissection or prosection, one of which is the concept of the somatic and visceral nervous systems peripheral structure and pathways. The objective of this study was to evaluate the use of drawing and labeling to gain appreciation of basic nervous systems concepts. Before the scheduled learning session, students were assigned a section to read in their textbook regarding the nervous system. At the beginning of the scheduled session, students were provided a sheet of paper with 4 boxes. In box 1 they were to copy a list of neuroanatomical terms that were shown on a Power Point slide. Next, they were shown on a Power Point slide an unlabeled drawing of the spinal cord and peripheral nerves. Using the terms listed in box 1, the various parts of the drawing were identified by the instructor for the students. The students were then asked to draw/sketch the drawing in box 2. In box 3, the students were asked to again copy the terms in box 1. In box 4, the students were asked to re‐draw/sketch the drawing in box 2 and label the drawing. When drawing and labeling in box 4, students were encouraged to check their neighbors’ drawing and labeling. Following this exercise, a shortened lecture was presented regarding the gross and cellular aspects of the neuroanatomical structures. This was followed by a Turning Point quiz using pictures from the textbook and lecture, and students were asked to check their answer with neighboring peers before submitting their answer. At the end of the neuroanatomical learning sessions, a survey with three questions using a five‐point scale was given to the students for them to evaluate the value for their learning of each segment (the list and drawing, lecture, and Turning Point quiz) of this learning session. Suggestions and comments were asked for. On a scale of 1–5, the value for each learning segment was as follows: list and drawing 4.3, lecture 3.8, Turning Point quiz 4.6. The survey results, and suggestions and comments, indicate that these various approaches to learning facilitate the learning process, and they serve as an active learning and peer teaching approach that is not boring.
Engineering Temperature Sensitive Alleles in Saccharomyces cerevisiae to Study the Genetic Interactions Between Ran point mutants and its Accessory Proteins
Authors: Tsepal Tenzin
Published: April 2020

Ran is an essential GTPase protein involved in bidirectional transportation between nucleus and cytoplasm. We engineered temperature sensitive (TS) mutant alleles of Ran accessory proteins in S. cerevisiae to study the genetic interactions. For essential proteins like Ran and its accessory proteins, TS mutants give insight into gene function without knocking out genes. By creating these TS mutant strains, we will be able to study the genetic interactions between Ran point mutants and TS mutant Ran accessory proteins to closely interrogate the biophysics and mechanism of the Ran GTPase cycle and it’s role nucleocytoplamsic transport.

Support or Funding Information

The 2019 NIH Summer Undergraduate Research Program at UMass Medical School. NIH Grant # R25 HL092610‐11