bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphine blocks spinal respiratory motor plasticity via mechanisms that require toll‐like receptor 4 signaling
Authors: Tadjalli Arash , Novak Jonathan , Bolser Donald C. , Levitt Erica S. , Mitchell Gordon S.
Published: April 2020

Opioid therapy is a common strategy for pain management; however, unfavorable side effects limit their clinical efficacy. Pro‐inflammatory cytokines oppose acute and chronic opioid‐induced analgesia and the ensuing inflammatory cascades that signal through toll‐like receptor 4 (TLR4)—pattern recognition innate immune receptors—are a significant contributor to some of the negative consequences of opioid therapy. While opioid‐induced inflammatory processes contribute to unwanted side‐effects (i.e. tolerance, dependence), it is unknown whether opioids, through pro‐inflammatory signaling, can affect the expression of plasticity within respiratory motor circuits. Adaptive plasticity within respiratory motor circuits is an important contributor to the preservation and/or restoration of respiratory function. For example, respiratory long‐term facilitation (LTF), a well‐studied model of respiratory motor plasticity, promotes breathing stabilization by increasing inspiratory drive to major breathing muscles. LTF is characterized by a long‐lasting compensatory increase in ventilation or respiratory motor nerve output triggered by exposures to acute intermittent hypoxia. Respiratory LTF is abolished by even a mild inflammation triggered by TLR4 receptor activation (e.g. via systemic lipopolysaccharides). Since opioid therapy activates TLR4 and induces neuro‐inflammation, we hypothesized that opioid delivery will abolish LTF through mechanisms that require TLR4 signaling. In adult Sprague Dawley rats, we show that acute systemic delivery of morphine, a prototypical opioid, blocks the expression of LTF in the phrenic motor system—a major motor pool that drives contractions of the diaphragm muscle. Furthermore, we show that morphine blocked phrenic LTF via mechanisms that require TLR4 signaling because systemic pre‐treatment with (+)‐naloxone—an opioid‐inactive TLR4 signaling inhibitor—restored phrenic LTF in morphine‐treated rats. Our findings suggest that loss of phrenic LTF following morphine treatment may remove an important stabilizing factor for breathing, potentially contributing to respiratory failure. Thus, attenuation of inflammatory signaling by opioid‐inactive compounds may help restore endogenous mechanisms of spontaneous plasticity within respiratory circuits, thereby minimizing opioid‐induced respiratory depression.

Support or Funding Information

NIH SPARC Initiative (OT2OD023854)
Role of AMPK in Adiponectin‐Mediated Restoration of Nitric Oxide‐Dependent Flow Induced Dilation in the Human Microvasculature
Authors: Cohen Katie E. , Schulz Mary E. , Gutterman David D. , Freed Julie K.
Published: April 2020

Flow‐induced dilation (FID), the ability of an arteriole to dilate in response to increased flow, is primarily mediated by endothelial‐derived nitric oxide (NO) in healthy individuals, however is replaced by hydrogen peroxide (H2O2) in patients with coronary artery disease (CAD). Our laboratory has shown that adiponectin restores NO‐mediated FID in microvessels from patients with CAD. Adiponectin activates multiple signaling pathways that promote NO formation, including activation of neutral ceramidase to hydrolyze ceramide, a sphingolipid that in elevated amounts promotes FID mediated by H2O2. Our prior work has shown that both NO and H2O2 contribute as mediators of FID in diseased vessels treated with adiponectin and ceranib‐1, an inhibitor of neutral ceramidase. As adiponectin is also capable of activating AMP kinase (AMPK) to increase cellular NO levels, we tested the hypothesis that AMPK activation is required to restore NO‐mediated FID in diseased arterioles. Human microvessels (80–200μm) were dissected from adipose tissue collected from patients diagnosed with CAD. Following cannulation and equilibration, vessels were constricted with endothelin‐1. Using videomicroscopy, vessel diameters were measured in response to graded increases in intraluminal flow. To investigate whether activation of AMPK is a key mechanistic step in adiponectin‐induced restoration of NO‐mediated signaling, vessels from patients diagnosed with CAD were first treated with the AMPK inhibitor Compound C (1μM, 4hrs) followed by adiponectin treatment (2μg/mL, 16–20hrs). Dilation to flow was maintained during exposure to the nitric oxide synthase (NOS) inhibitor L‐NAME (100μM) (69.8% max dilation±2.7, n=2) as well as in the presence of the PEG‐Catalase (500U) (64.5%±21.2, n=4) compared to compound C and adiponectin alone (61.0%±9.4, n=4). To determine whether ceramide hydrolysis via neutral ceramidase contributes to the maintained dilation despite NOS inhibition, FID was also assessed in diseased arterioles treated with both compound C and ceranib‐1 prior to treatment with adiponectin. Both NO and H2O2 contributed to dilation under both AMPK and neutral ceramidase inhibition as dilation was observed in the presence of L‐NAME (33.6%±15.9, n=3) or PEG‐Catalase (51.7%±23.9, n=3). However, FID was decreased during exposure to both L‐NAME and PEG‐Catalase (11.6%±12.4, n=2). These preliminary data suggest that adiponectin promotes NO formation through multiple pathways and contributes to the redundancy of endothelial‐derived mediators in order to maintain FID.

Support or Funding Information

This research was supported by National Institute of Health (NHLBI) K08HL141562 (J.K.F.) and National Institute of Health 5R38HL143561‐02 (M.E.W.).
Activation of VTA NtsR1 Neurons Promotes Weight Loss Behaviors
Authors: Perez-Bonilla Patricia , Santiago-Colon Krystal , Matasovsky Jillian , Leinninger Gina M.
Published: April 2020

Dopamine (DA) neurons in the ventral tegmental area (VTA) modulate motivation to engage in physical activity and food intake, dual behaviors that are disrupted in obesity. However, the functional heterogeneity of VTA DA neurons, and lack of molecular markers to distinguish them, has prevented determination of which DA neurons might be leveraged to support weight loss behaviors. We identified a distinct subset of VTA DA neurons defined by their expression of neurotensin receptor‐1 (NtsR1) and we hypothesized that these “VTA NtsR1 neurons” modulate feeding restraint and physical activity that could support weight loss. To test this hypothesis, we expressed Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in the VTA NtsR1 neurons of normal weight and diet‐induced obese mice, permitting in vivo activation of VTA NtsR1 neurons by treatment with the DREADD‐ligand clozapine‐N‐oxide (CNO). Acute activation of VTA NtsR1 neurons (24hr) in normal weight and diet‐induced obese mice significantly decreased body weight by reducing food intake and increasing physical activity. Moreover, the weight loss of diet‐induced obese mice was maintained with daily activation of VTA NtsR1 over 7d. Intriguingly, activation of VTA NtsR1 neurons also suppressed how much mice would work to obtain sucrose rewards even when there was high motivation to consume (e.g. in fasted mice or diet‐induced obese mice.) These data suggest that VTA NtsR1 neurons may be useful to support weight loss behavior. Yet, the translational potential of NtsR1 was previously dismissed due to the broad expression of NtsR1 throughout the brain and periphery, and data that systemic NtsR1 agonists cause adverse hypothermia and vasodepressor responses. However, we found that specifically activating VTA NtsR1 neurons does not alter blood pressure, core body temperature or anxiety behavior. Thus, activation of VTA NtsR1 neurons during states of high‐appetitive drive such as fasting and obesity promotes dual behaviors that support weight loss without causing adverse cardiorespiratory effects or stress responses. In the future, strategies to selectively modulating VTA NtsR1 neurons could provide a safe, effective avenue for weight loss.

Support or Funding Information

Supported by funding from the National Institutes of Health to Perez‐Bonilla (T32GM092715) and Leinninger (R01‐DK103808)
Effect of hyperglycaemia on the growth of lung pathogens in Artificial Sputum Media
Authors: Vasiljevs Stanislavs , Gupta Arya , Baines Deborah
Published: April 2020

Elevated glucose concentration in the airway surface liquid due to high blood glucose (hyperglycaemia) has been associated with the increased risk of developing pulmonary infection. It has recently been shown that lungs are colonisedby resident bacteria and are not truly sterile. Several chronic lung conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), have been shown to induce changes in this bacterial population.

Here we sought to develop an in vitro model using artificial sputum media (ASM) to investigate the effect of hyperglycaemia on Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA), two of the most common pathogens in CF patients.

SA (ATCC29213) and PA (H174) were grown in the artificial sputum media (ASM) at varying glucose concentrations. Approximately 3×105 bacteria were added to 15 mL ASM and grown at 37°C with constant shaking. Two‐way ANOVA with Geisser‐Greenhouse correction and Pearsons correlation coefficients were used for statistical analysis.

ASM supplemented with 4 mM glucose had a significant effect on the growth rate of both S. aureus and P. aeruginosa when compared with ASM with no glucose (p = 0.0028, n=5 for SA and p < 0.0001, n=5 for PA). Compared with growth in Mueller Hinton broth, only P. aeruginosa exhibited increased growth rate in the presence of 4 mM glucose (p = 0.0082, n = 3). Growth of both, S. aureus and P. aeruginosa, was correlated with the depletion of glucose in the media (p = 0.0002, n=5 for PA and 0.0038, n=5 for SA). The production of lactate by S. aureus was not related to the depletion of glucose as concentrations for both reached zero at the sixth hour of growth.

As ASM mimics the nutrients available in the lungs, it facilitates the study of lung pathogens in the presence of glucose. The established in vitro assay in this study can be further utilised to examine the competition and/or co‐existence between multiple pathogens that are known to reside within the lungs.

Support or Funding Information

Research funded by MRC‐DTP
Development of New Methods for Enhanced Conformational Sampling of GPCRs
Authors: Serrano Erik , Abrol Ravinder
Published: April 2020

G protein coupled receptors (GPCRs) are integral membrane proteins that enable a cell to respond to extracellular stimuli like light, small molecules, peptides, and proteins. These receptors have evolved as highly flexible proteins that possess multiple functionally important conformational states, which enable these receptors to activate pleiotropic signaling events inside the cells. Recent progress in membrane protein structural biology techniques and cryo‐electron microscopy is providing structural evidence of two or more distinct receptor conformations for these receptors. Computational structural modeling approaches can complement and supplement these studies by predicting all functionally important states of a receptor, however, they run into the classical protein conformational sampling problem. We are developing a computational biophysical method called Enhanced Conformational Markov‐state Sampling in Membrane BiLayer Environment (EnCoMSeMBLE) to solve this sampling problem for GPCRs. We are utilizing Markov‐State‐Models to combine the Boltzmann sampling of receptor conformations from molecular dynamics (MD) with the brute‐force sampling of receptor conformations from our previously developed ActiveGEnSeMBLE method. The method was applied to the A2A receptor starting from the active state by only using traditional MD simulations. This is not surprising because the pre‐activce states is a downhill direction on the energy landscape. However, applying both MD and ActivateGEnSeMBLE methods was able to predict pre‐active states from the inactive states which, classical MD cannot achieve. Our results indicated that our methods provide an unbiased method to sample the conformational landscape. This can protentional sample unexplored regions of the conformational landscape and find novel conformations that can elucidate the mechanistic properties that GPCRs possess.

Support or Funding Information

This project was funded through a grant from the National Institutes of Health (NIH) Building Infrastructure Leading to Diversity (BUILD) #5TL4GM118977 or # 5RL5GM118975.
Single‐Molecule Optical Replication Mapping (ORM) Suggests Human Replication Timing is Regulated by Stochastic Initiation
Authors: Rhind Nick , Wang Weitao , Klein Kyle , Proesmans Karel , Yang Hongbo , Hastie Alex , Yue Feng , Bechhoefer John , Chunlong Chen , Gilbert David
Published: April 2020

DNA replication timing is regulated by the timing of initiation across the genome. However, there is no consensus as to how initiation timing is regulated. Deterministic models contend that different initiation sites are programed to initiate at different, well‐defined times. Stochastic models posit that different initiation sites have different initiation probabilities but can fire at any time during S. Sites with a high probability of initiation are more likely to fire early and thus will, on average, initiate early in S. Ensemble replication timing assays that report only average initiation time cannot distinguish between these two types of models.

To test if replication initiates deterministically or stochastically, we developed Optical Replication Mapping (ORM) a high‐throughput, genome‐wide, single‐molecule replication mapping approach that combines in vivo replication labeling with the Bionano genomic mapping technology. We have mapped replication in over 20 human cell datasets, representing over 2000‐fold coverage of the human genome, allowing us to map sites of replication initiation that are used in fewer than 0.1% of S phases.

Our analyses support three major conclusions. 1) Replication initiates at many infrequently‐used initiation sites spread across initiation zones of 30 to 100 kb. We find no evidence for discrete, high‐frequency initiation sites in the human genome. These result suggest that "replication origin", in the sense of a defined genetic element, is not a useful concept in metazoans. 2) Initiation zones correlate well with regions of DNase I accessible DNA, such as promoters. G4 sequences do not appear to be predictive of sites of initiation, beyond their enrichment in DNase I accessible chromatin. These results suggest that a major driver of initiation probability is DNA accessibility. 3) We observe a low frequency of early initiation across late‐replicating regions, and these initiation events are enriched in late initiation zones. Observed initiation rates across all regions of the genome are consistent with a stochastic mechanism of replication timing and inconsistent with a deterministic replication timing program. We propose that the dominant parameter that regulates replication timing is the initiation probability across initiation zones, with a high degree of heterogeneity of initiation sites within these zones, and no spatial or temporal coordination between individual initiation events.
Comparative In Vitro Nephrotoxicity of Three 3,5‐Dihaloanilines in Isolated Renal Cortical Cells from Male Fischer 344 Rats
Authors: Rankin Gary , Hicks William M. , Ju Jae , Vidrine David , Racine Christopher , Anestis Dianne
Published: April 2020

Halogenated anilines are widely used to manufacture dyes, pesticides, pharmaceuticals and industrial products. Toxicity induced by these compounds includes hepatotoxicity, hematotoxicity, splenotoxicity, immunotoxicity and nephrotoxicity. We have shown that among the mono‐ and dichloroanilines, 3,5‐dichloroaniline (DCA) is the most potent nephrotoxicant in vivo and in vitro. DCA nephrotoxicity in vitro is attenuated by primary antioxidants (α‐tocopherol, ascorbate) and antioxidant nucleophiles (glutathione, N‐acetyl‐L‐cysteine), suggesting a role for free radicals in DCA nephrotoxicity. The purpose of this study was to compare the nephrotoxic potential of DCA with the nephrotoxic potential of 3,5‐dibromo‐ and 3,5‐diiodoaniline (DBA and DIA, respectively) in isolated renal cortical cells (IRCC) from male Fischer 344 rats. The comparative effect of antioxidants on 3,5‐dihaloaniline (DHA) nephrotoxicity was also determined. IRCC (~4 million cells/ml; 3 ml) were incubated with shaking at 37°C under a 95% oxygen/5% carbon dioxide atmosphere with a DHA (0.25 – 1.0 mM) or vehicle (dimethyl sulfoxide) for 15–90 min with or without a pretreatment. General cytotoxicity was determined by assessing trypan blue exclusion of IRCC and measuring changes in lactate dehydrogenase (LDH) release. Among the three DHAs, DBA was the most potent nephrotoxicant, inducing LDH release at 0.25 mM by 30 min. DCA was the least potent nephrotoxicant, requiring 90 min at 0.5 mM exposure to induce cytotoxicity, while DIA was intermediate in potency by inducing cytotoxicity at 60 min with 1.0 mM exposure. Pretreatment with an antioxidant (α‐tocopherol, ascorbate) or antioxidant/nucleophile (glutathione, N‐acetyl‐L‐cysteine) attenuated cytotoxicity induced by the three DHAs (1.0 mM). These results indicate that the decreasing order of nephrotoxicity in IRCC was DBA > DIA > DCA, and that free radicals play a role in the DHA‐induced nephrotoxicity induced by the three compounds tested.

Support or Funding Information

Supported in part by NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence.
Nef Dimerization is Required for Antagonism of the Host Cell Restriction Factor, SERINC5
Authors: Staudt Ryan P. , Smithgall Thomas E.
Published: April 2020

HIV‐1 Nef is a virulence factor that is essential for immune escape of HIV‐infected cells, viral pathogenesis and AIDS progression. Nef lacks intrinsic biochemical activity, functioning instead by interacting with host cell effector proteins to hijack multiple signaling and endocytic pathways. Many Nef functions depend on dimerization of the protein, including enhancement of viral infectivity, replication, and immune cell receptor downregulation. Work in our lab, through both X‐ray crystallography and virological assays, has shown that distinct hydrophobic residues (L112, Y115, F121) are key in Nef dimer formation, and disruption of these residues significantly impairs many Nef functions. Pharmacologic disruption of the Nef homodimer may therefore represent a novel approach to antiretroviral therapy.

Here we investigated whether Nef dimerization is required for antagonism of the host cell restriction factor SERINC5 (S5). S5 is a potent suppressor of HIV‐1 infectivity that is antagonized by Nef via binding and downregulating S5 from the surface of infected cells, preventing its incorporation into nascent virions. For our studies, HIV‐1 virions were produced in 293T cells transfected with the HIV‐1 provirus and S5, Jurkat WT and S5‐knockout cells, or donor PBMCs (which naturally express high levels of S5). Viral supernatants were harvested from these “producer” cells and normalized for p24 AlphaLISA. Infectivity of producer‐cell viruses was assessed using TZM‐bl reporter cells, which couple the HIV‐1 LTR to expression of luciferase. Mutation of conserved Nef residues essential for homodimerization (L112A/D, Y115D, F121A) reduced the infectivity of HIV‐1 produced from all three producer cell lines in a S5‐dependent manner. S5 incorporation into virions was increased in the presence of Nef dimer interface mutants, suggesting that dimerization is important in its ability to exclude S5 from newly synthesized virions. Recruitment, downregulation, and endocytic trafficking of S5 by wild type and dimerization‐defective Nef mutants were also examined via confocal microscopy. Nef dimer interface mutants also failed to induce colocalization of S5 with Rab7+ late endosomes, suggesting that Nef dimerization is essential for S5 internalization and degradation via the lyso‐endosomal pathway. By contrast, Nef mutants retained their ability to interact with both S5 and subunits of the AP‐2 clathrin adaptor protein essential for S5 endocytosis, as shown via bimolecular fluorescence complementation. Taken together, these results demonstrate that downregulation of S5 and subsequent enhancement of viral infectivity requires Nef homodimerization, providing further support for pharmacological targeting of Nef quaternary structure in HIV‐1 cure strategies.
Matrix Metalloproteinase 9 (MMP9) Inhibitor Prevents Mustard Induced Microvesication and Promotes Cutaneous Wound Healing
Authors: Chang Yoke-Chen , Muhn Selina , Hahn Rita A. , Gordon Marion K. , Gerecke Donald R.
Published: April 2020

Skin exposure to the chemical warfare agent, sulfur mustard (bis‐2‐chloroethylsulfide, SM), is characterized by severe blister formation and a prolonged inflammatory response. The mechanisms of these SM toxic effects are still poorly defined, and effective medical countermeasures against SM‐induced cutaneous injury have yet to be established. Animal models demonstrate disruption of the skin architecture at the dermal‐epidermal junction (DEJ) when exposed to the vesicant, SM; the damage may be further exacerbated by overexpression of MMP9. Studies show MMP9 degrades basement membrane (BM) components and regulates inflammation in tissue injuries; inhibition of MMP9 suppresses inflammation and promotes wound repairs in tumors and various wound models. In the present studies, ([N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide], BiPS), a specific MMP9 inhibitor, was evaluated for its ability to prevent microvesication, suppress inflammation, and promote wound healing following SM exposure using the mouse ear vesicant model. Treatment of male CD1 mouse ear skin with SM (0.08 mg) caused a characteristic SM induced injury including edema, inflammatory cell infiltration, and the formation of microvesicles at the DEJ. Expression of MMP9 mRNA and protein in the skin progressively increased with time 24–168 h following SM exposure. Immunofluorescence studies showed disruption of the BM molecule type IV collagen (COL4), increased expression of inflammation marker, COX2, and upregulation of the skin wound marker keratin 6 (K6) in SM induced skin wounds. SM also caused macrophage (F4/80) accumulation in the tissue which persisted up to 168 h post exposure. Pretreatment of ear skin with BiPS significantly reduced SM‐induced dermal edema and maintained the integrity of the DEJ‐BM as evidenced by contiguous expression of COL4. At 168 h post SM exposure, mRNA and protein expression of MMP9 was significantly downregulated in BiPS treated mice. BiPS treatment also reduced the number of macrophages accumulating in the tissue, and suppressed expression of COX2, and downregulated expression of K6. Taken together, these data indicate that BiPS treatment was able to suppress both inflammation and degradation of BM zone in skin due to SM. This compound was effective in targeting MMP9 to protect against SM induced microvesication and promote wound repairs. MMP9 inhibitors may be useful as medical countermeasure against SM‐induced cutaneous injury.

Support or Funding Information

Supported by NIH grants ES005022 and AR055073.
Cisplatin Decreases ENaC Activity Contributing to Renal Sodium Wasting
Authors: Soares Antonio Garcia , Mironova Elena V. , Abd El-Aziz Tarek M. , Stockand James D.
Published: April 2020

Cisplatin (CDDP) is a platinum‐containing compound that has antineoplastic effects due to its ability to bind DNA and inhibit replication. While an important chemotherapy drug, CDDP results in a renal salt and water‐wasting syndrome and is nephrotoxic. The origin of the renal salt and water‐wasting caused by CDDP is obscure; though, some evidence suggests that broad inhibition of the Na+/K+‐ATPase by CDDP results in generalized decreases in tubular reabsorption causing increases in sodium and water excretion. The effects of CDDP on the epithelial Na+ channel (ENaC), which is the final arbiter of renal Na+ excretion, are unknown. We test here if CDDP affects the activity of ENaC to influence renal salt and water excretion. These studies focus on understanding the cellular mechanism by which CDDP may affect ENaC. For these studies, C57Bl/6 male mice were randomly divided into 3 groups, as follow: 1) Saline, 2) CDDP and 3) Benzamil (BZM) treated. For metabolic cages experiments, C57Bl/6 male mice received a Na+ load (100 μL of a 0.9% NaCl solution) and were treated with CDDP (25 mg/kg, i.p.) or BZM (1.4 mg/kg, i.p.). Then, water consumption, Na+ intake, urine volume, urinary Na+ and creatine excretion were evaluated every 2 h over a 10 h period. To directly quantify ENaC activity in these animals, principal cells were patch‐clamped in split‐open tubules using the cell‐attached configuration. After recording baseline ENaC activity for 5 min in these tubules, they were treated with CDDP (0.5 to 1.5 μM) with ENaC activity quantified in the presence of this drug over the next 5 min. Preliminary results show that CDDP increases excretion of a Na+ load, increases fractional excretion of sodium (FeNa) and decreases ENaC activity in isolated tubules.
Importantly, our studies show that, unlike ouabain, CDDP does not affect channel conductance in cell‐attached patches. Such observations are most consistent with CDDP not affecting the electrochemical forces driving Na+ transport across principal cells, which would change in response to changes in pump activity, but rather decreasing ENaC activity through direct effects on the channel or a channel regulator. In conclusion, these observations are consistent with CDDP actions on ENaC, independent of effects on the pump, contributing to the renal salt and water wasting caused by this chemotherapy drug.

Support or Funding Information

This research was supported by NIH/NIDDK grants R01DK113816 and R01DK117909.
Characterizing a Phosphoglycolate Phosphatase Ortholog in D. melanogaster
Authors: Dwyer William Pascal
Published: April 2020

Phosphoglycolate phosphatase (PGP) is an important metabolic enzyme found in prokaryotes and in many higher eukaryotes. In mammals, PGP has been identified as a key regulator of sugar and fat metabolism and as a ‘guardian angel’ enzyme which dephosphorylates toxic glycolytic side products. We aim to elucidate the role of PGP in fruit fly development and overall energy metabolism by characterizing mutants lacking CG5567, a recently discovered ortholog of PGP in the fruit fly D. melanogaster. We predicted that loss of CG5567 would decrease glycolysis through a build‐up of inhibitory side products. Using varying concentrations of sugar in the diet, we modulated flux through glycolysis and assessed both larval and adult mutant phenotypes through a series of behavioral and metabolic assays. Additionally, we assessed genotypic influence on larval aerobic glycolysis by respirometry using an adapted portable photosynthesis system. Preliminary results indicate both genotype and diet separately influence levels of triacylglycerides (TAG) in D. melanogaster and have a synergistic impact on female TAG concentration. Consistent with previous findings that CG5567 is a non‐essential gene (in contrast to its mouse ortholog), we found no increased mortality or delayed development in PGP‐mutant larvae reared on a high‐sugar diet. However, our results suggest PGP is implicated in adult fly weight and energy output, thus confirming a conserved role for PGP in regulating metabolism.
A potential role of Zfra in mitigating traumatic brain injury transition to Alzheimer’s disease‐like symptom in mice
Authors: Liu Tsung-Yun , Chang Nan-Shan
Published: April 2020

Most recently, tumor suppressor WWOX is defined as a risk factor for Alzheimer’s disease(AD). Previously, we determined that accumulation of pS14‐WWOX in the brain hippocampus and cortex correlates with the occurrence of AD and probably other neurodegeneration. Zinc finger‐like protein that regulates apoptosis (Zfra) suppresses pS14‐WWOX accumulation and restores memory loss in triple transgenic mice (3xTg) for AD. During the acute phase traumatic brain injury (TBI), overly expressed WWOX becomes phosphorylated at Tyr33 and is accumulated in the nuclei to cause neuronal death in the impacted areas. We determined that in the novel object recognition tests, mice surviving from TBI (2.8 to 3.3 Newtonian force) significantly spent less exploring time on new objects in both long‐term and short‐term memory tests post TBI for 18 weeks, indicating the occurrence of damage in long‐ and short‐term memory. Further, control and TBI mice were subjected to Morris water maze tests post TBI for 10 weeks to determine the hippocampus‐dependent, spatial memory. Both groups showed a time‐dependent decrease in escape latency, indicating that mild TBI didn’t affect the learning capabilities. BALB/c mice received Zfra4‐10 peptide via tail vein injections prior to TBI (1.7 Newtonian force). Two weeks later, these treated mice significantly spent more time on probing the new object in the short‐term memory task compared to control mice, suggesting that Zfra peptide prevents TBI‐induced memory loss. Interestingly, by immunohistochemistry, significant upregulation of tau phosphorylation at T181 and amyloid beta aggregates was shown rapidly in death‐inducing impact in BALB/c mice (3.5 Newton), and that Zfra significantly prevented the upregulation (~20 to 33%). Together, our observations suggest the transition of TBI to AD‐like symptom in mice are likely to occur, and that Zfra4‐10 peptide prevents the transition.

Support or Funding Information

Ministry of Science & Technology, Taiwan, National Health Research Institute, Taiwan, Department of Defense, USA
Hepatocyte HIF‐1alpha mediates obesity‐induced decreased incretin effects
Authors: LEE Yun Sok
Published: April 2020

The decrease in incretin effects is an important etiologic component of type 2 diabetes with unknown mechanisms. In an attempt to understand obesity‐induced changes in liver oxygen homeostasis and its metabolic consequence, we came across a novel finding that liver HIF‐1α expression was increased mainly by soluble factors released from obese adipocytes, leading to decreased incretin effects. In‐depth mouse metabolic phenotyping revealed that obesity increased first‐pass degradation of an incretin hormone GLP‐1 with increased liver DPP4 expression and decreased sinusoidal blood flow rate, reducing active GLP‐1 levels in peripheral circulation. Deletion of hepatocyte HIF‐1α KO blocked these changes induced by obesity: hepatocyte HIF‐1α KO mice were protected from obesity‐induced glucose intolerance without changes in body weight, liver steatosis, or insulin resistance. Deletion of hepatocyte HIF‐2α did not change liver DPP4 expression but improved hepatic steatosis. Our results identify a previously unknown pathway for obesity‐induced impaired beta cell glucose response (incretin effects) and the development of glucose intolerance through inter‐organ communications.
Hemodynamic Sex‐differences in Diastolic Function
Authors: Ogilvie Leslie M. , Edgett Brittany A. , Huber Jason S. , Platt Mathew J. , Brunt Keith R. , Simpson Jeremy A.
Published: April 2020

Background

In the past decade, cardiac research has focused on the emerging crisis of diastolic dysfunction, commonly observed in patients with heart failure with preserved ejection fraction (HFpEF) — a disorder with ineffective management strategies. Diastolic dysfunction is abnormal active relaxation and/or passive stiffness of the ventricle. Because echocardiography is non‐invasive and accessible to more specialities, it remains widely used in the diagnosis and prognostic gradation of diastolic dysfunction. However, echocardiography provides indirect estimations of diastolic indices because diastolic measures were calibrated based on direct measures of end diastolic filling pressure (the gold standard for diastolic assessment). As it is increasingly evident that the manifestation of heart failure in men/males and women/females is not the same, a focus on sex‐specific mechanisms and physiology of diastole is warranted.

Experimental Approach

The primary objective of this work is to perform a comprehensive hemodynamic analysis of diastolic function in males and females to highlight sex‐differences that exist in diastolic physiology and provide implications in cardiac pathology. Male and female Wistar rats were anesthetized with isoflurane and a pressure catheter was inserted into the right carotid artery and advanced into the left ventricle. Pressure measurements were sampled at 2kHz and digitized using custom software. Diastolic parameters – end diastolic pressure (EDP), maximum relaxation rate (dP/dtmin), isovolumetric relaxation time constant (Tau Weiss, Glantz, Logistic) – were analyzed to evaluate diastolic function.

Results and Conclusions

Diastolic function in healthy males and females is not equal, suggesting there are sex‐specific differences in diastolic physiology. In a pathological state, each of the diastolic parameters follows its own unique time course until severe dysfunction is present, suggesting that diastole cannot be reconciled by any one parameter. EDP, dP/dtmin and Tau Logistic should be used together to stratify dysfunction as diastole is a dynamic process and each parameter captures a specific nuance.

Support or Funding Information

Canadian Institutes of Health Research
Cardiac Regeneration Occurs Following a Puncture Wound to the Heart of the Leopard Gecko (Eublepharis macularius)
Authors: Jacyniak Kathy , Vickaryous Matthew Kenneth
Published: April 2020

Survivable injuries to the heart resolve via one of two outcomes: scar formation or tissue regeneration. Among mammals, injury to the heart typically results in the loss of contractile muscle cells (cardiomyocytes) and the formation of a non‐contractile fibrous scar. In contrast, some teleost fish and salamanders are capable of tissue‐specific regeneration, thus replacing lost or damaged cardiomyocytes. Here, we investigate the regenerative capacity of the reptilian heart, focusing on the lizard Eublepharis macularius, the leopard gecko. Unlike mammals, but similar to teleost fish, the gecko heart has a single ventricle. To determine if the ventricle was capable of regeneration, we performed a cardiac puncture (cardiocentesis) – a penetrating wound that passes through the thoracic cavity and into the heart. Cardiac punctures are readily tolerated by geckos, and normal (pre‐injury) behaviours are observed within hours of the procedure. To characterize the reparative events, we used serial histology and immunostaining for markers of cell proliferation (proliferating cell nuclear antigen, PCNA) or cell death (Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]), each with markers for cardiomyocytes (myosin heavy chain [MHC], alpha‐smooth muscle actin [α‐SMA]), and fibroblasts/endocardial cells (Vimentin). Prior to injury, we observed proliferating populations of both cardiomyocytes and non‐cardiomyocytes. One day post‐cardiac puncture (dpc), the wound site is characterized by cell death, a localized loss of cardiomyocytes, and the formation of a blood clot capping the puncture. Between 5 and 10 dpc, there is an increase in the number of proliferating cardiomyocytes bordering the lesion, simultaneous with an increase in proliferating non‐cardiomyocytes (cardiac fibroblasts and endothelial cells) within the wound bed itself. By 14dpc, mature cardiomyocytes have repopulated the wound site, restoring the original architecture of the myocardium. Overall, our findings reveal that regeneration of the gecko heart closely resembles that of zebrafish, and is distinct from the scar‐forming response of mammals.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants 400358
The Study of Sphingolipids Metabolism and Its Effect on Brain Disorders
Authors: Hernandez Ricardo
Published: April 2020

Sphingolipids are molecules involved in important processes like cell growth, signaling, cell structure and apoptosis. It is known that sphingolipids metabolism is involved in the proper development of the brain and deficiency in sphingolipids homeostasis lead to psychiatric disorders Sphingolipids are also known to play a role in other disorders, such: necrosis. In this research, sphingolipids metabolism is studied using Saccharomyces cerevisiae as a model. A CSG2 deletion mutant, accumulates inositolphosphoryl ceramide (IPC), the first complex sphingolipid. When it is exposed to nitrogen starvation conditions with a fermentable carbon source present, the cells do not adapt properly to this stressor because the mitochondria cannot increase the MMP (mitochondria membrane potential), which leads to an increase in reactive oxygen species (ROS) and cell death. Although, when the same mutant is exposed to nitrogen starvation conditions with a nonfermentable carbon source present, cells grow normally. Cells are rescued by the ISC1 gene activity. ISC1human’s homologue gene codes for the protein sphingomyelinase. This protein breaks down complex sphingolipids into ceramide. It is well known that ISC1 is involved in process related to nutrient availability and it is activated under nonfermentable carbon sources. Currently, Isc1p is the only protein known that can break down complex sphingolipids in yeast. This organism has two main types of complex sphingolipids, mannosylated and non‐mannosylated. In this research, we do not only study IPC (non‐mannosylated complex sphingolipid) but also the other two more complex mannosylated sphingolipids: mannose‐inositol‐phosphoceramide (MIPC) and mannose‐(inositol‐P)2‐ceramide (M(IP)2C). It has been shown that Ipt1D, mutant that accumulates MIPC since, the gene, IPT1, that codes for the protein that turns MIPC into M(IP)2C has been deleted, survive under nitrogen starvation when a fermentable carbon source is present. Our results suggest that Isc1p cannot bind to complex sphingolipids when a mannose group is attached to them and another protein, not known yet, that carries out the function of breaking down mannosylated complex sphingolipids when a fermentable carbon source is present. Therefore, this research focuses on the study of sphingolipids metabolism and homeostasis, studying genes that are involved in the anabolism as well as the catabolism pathways. This research uses data collected form hands‐on experiment, for instance: TLC, fluorescence microscope, cell viability assay; and from computational methods. The results will lead to a better understanding of sphingolipids homeostasis and can help to target the treatment of disorders caused by these molecules.

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Presbyterian College, Chemistry and Biochemistry Department
Calcium Binding Preferentially Stabilizes Apical End of Dysferlin C2A
Authors: Otto Shauna , Kumar Tanushri , Reardon Patrick , Johnson Colin
Published: April 2020

Dysferlin is a crucial component of the eukaryotic membrane repair machinery, helping to seal microtears in the sarcolemmal membrane that arise from constant mechanical insult. Loss of this protein results in several muscle‐wasting phenotypes in both human patients and mouse models. Dysferlin contains seven C2 domains, which are well‐documented Ca2+‐dependent lipid sensors, which suggests a putative role in the calcium‐sensitive steps of membrane repair for dysferlin. A deeper understanding of the mechanisms that govern how dysferlin coordinates calcium signals with repair machinery mobilization is essential to develop therapeutic strategies.
Out of the many C2 domains, the N‐terminal C2A domain is of particular interest because it has proved essential to the membrane repair process. Initial thermodynamic studies of this domain showed a
curiously low energy of unfolding, suggesting that the domain is partially unfolded under resting calcium conditions. Here we use nuclear magnetic resonance spectroscopy and supporting biophysical techniques to examine the stabilizing effect of millimolar levels of calcium at the residue level and conclude that calcium binding primarily affects the apical end of the C2A domain of dysferlin. Calcium binding to C2 domains has been previously shown to induce subtle conformational shifts towards a more membrane‐competent fold, however, our studies provide the
most drastic example of this phenomenon to date.
Providing Authentic Research Opportunities In A Large Laboratory Course By Studying Metabolic Channeling Properties Of Malate Dehydrogenase Isoforms In Trypanosoma Brucei
Authors: Springer Amy
Published: April 2020

We are using a Course‐Based Undergraduate Research Experience (CURE) model to provide opportunities for Biochemistry and Molecular Biology (BMB) majors to gain exposure to laboratory research. Upper class undergraduates perform a four‐week “boot camp” of common laboratory techniques and then work, as teams, on projects related to a faculty member’s research. Proejcts focused on exploring interactions between Malate dehydrogenase (MDH) with citrate synthase or fumarate hydratase, adjacent enzymes in the tricarboxylic acid (TCA) cycle. Recent studies from some plant and mammalian species suggest that some MDH isoforms form specific interactions with these enzymes to promote high flux of intermediates via substrate channeling. We are interested in metabolic regulation in Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness, this unicellular eukaryote is dependent on metabolic regulation for successful colonization of the Tsetse fly and subsequent infection of mammalian hosts. Growth within the fly utilizes oxidative phosphorylation of amino acids, whereas growth in the mammalian bloodstream is strictly glycolytic. Three MDH isoforms exist in T. brucei, localized to mitochondria, cytoplasm and glycosomes, respectively. We are interested in determining if any of the T. brucei MDH isoforms interact with other metabolic enzymes as appropriate to promote the proper metabolic program. We have used student projects to generate clones for recombinant expression of all three trypanosome MDHs, citrate synthase (CS) and both fumarate hydratase (FH) isoforms. While some student teams design projects de novo, others choose to continue a project from a previous semester. Student teams have conducted expression studies, interaction assays and enzymatic analyses to characterize these enzymes. Students give presentations, a final poster and write and revise reports and a research proposal. Student also share their ideas and projects with other members of a consortium of faculty from other institutions in the protein‐centric MDH CURE community (MCC), working on similar projects with other organisms. MCC provides a wealth of possibilities for comparative studies and to offer faculty and students access to scientific information and community beyond the students and instructors in the course, assisting development of hypotheses and sharing of reagents.
Ferric Chelate Reductase Knockdown in Drosophila S2 Cells
Authors: Murphy Laura Grace , Holst Jessica , Kane Gregory , Gorman Maureen J. , Ragan Emily J.
Published: April 2020

Although the mechanisms of cellular iron uptake in mammals are largely understood, there is still little known about the mechanism of iron transport in insects. Not only is iron essential, it can also be toxic and therefore needs to be well regulated to maintain balance between necessity and toxicity. In mammals, a duodenal cytochrome b acts as a ferric reductase to allow for uptake of iron into intestinal cells. We identified a cytochrome b561 family member in Drosophila, CG8399, that is expressed in Drosophila S2 cells. CG8399 is similar to ferric chelate reductase‐1 in humans as both contain reeler, domon and cytochrome b561 domains. We are testing the hypothesis that there is reduction of iron by CG8399, which allows the transport of iron into the cells. To test this hypothesis, we used RNA interference (RNAi) to knockdown the CG8399 mRNA. We quantified the RNAi knockdown caused by RNAi using real time PCR and compared iron concentrations between untreated and treated cells using a ferrozine‐based iron content assay. A lower concentration of iron in CG8399 RNAi‐treated cells would support our hypothesis.

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NSF IOS‐1656407
Designing a Measurement Methodology for the Upper Aerodigestive Tract of Phoca vitulina
Authors: Nourbakhsh Hirad , Adams Arlo , Vogl Wayne , Dawson Camilla , Raverty Stephen , Haulena Martin , Skoretz Stacey A.
Published: April 2020

In North America, wild harbor seal neonates and pups (Phoca vitulina) are among the most common marine mammals admitted to rescue centers. Nutritional support is typically via gavage as most are admitted prior to weaning. In order to advance seal feeding and swallowing rehabilitation, we require a comprehensive understanding of their upper aerodigestive tract (UAT) as it applies to swallowing physiology. Anatomical studies of the harbor seal UAT are limited, and standardized UAT measurements for neonates and pups have not yet been determined. Our objective is to design a systematic method to measure their UAT structures and then obtain quantitative values. During post mortem examination, we photographed UAT structures using scalars. Structures included: tongue, vocal folds, cricoid cartilage, trachea, and epiglottis. A priori, we developed a manual outlining measurement start and endpoints using ImageJ (NIH, 2010). Following measurements, we summarized results with means (standard deviations, [SD]) and medians (interquartile ranges, [IQR]) as appropriate and explored correlations (Pearson’s correlation coefficient, one‐tailed) between UAT structures and other body measurements. We used IBM SPSS analytical software version 26 (IBM Corporation, 2019) for analyses with significance of p<.05. Our specimens included 14 deceased harbor seal neonates or pups (9 males, 5 females) aged between three days and 2 months old at the time of death. Causes of death were either: spontaneous, congenital, or due to infectious, metabolic or malnutrition disorders, or by euthanasia after a humane endpoint was reached. On admission to rehabilitation, body measurement ranges included: 46.0 to 57.0 (body length [cm]), 36.0 to 59.0 (axillary girth [cm]) 5.8 to 12.6 (weight [kg]). Post‐mortem UAT measurements (mm, mean ± SD) included: 1) tongue tip to epiglottis base: 82.7 ± 6.9, 2) vocal fold length: 13.9 ± 2.0, 3) tracheal diameter: 10.3 ± 1.4, and 4) epiglottic length: 20.0 ± 2.8. Correlations between seal body length and UAT dimensions were not statistically significant. In conclusion, we have developed a systematic method to measure harbor seal neonate and pup UAT structures. To the best of our knowledge, these measurements, as they relate to swallowing, have not been previously reported. These findings expand the seal macroscopic anatomy knowledge base and contribute to cross‐species conceptual convergences between human and seal swallowing physiology and its rehabilitation.

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Supported by a UBC Faculty of Medicine Summer Student Research Program 2019 award to Hirad Nourbakhsh, UBC Faculty of Medicine Start‐up grant to Stacey A. Skoretz and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to A. Wayne Vogl (RGPIN‐2018‐03727).