bluuebunny/crossref_metadata_2025_split huggingface dataset:
Skeletal Muscle Mitochondrial Capacity in Patients with Statin‐Associated Muscle Symptoms (SAMS)
Authors: Mangone Laura , Parker Beth A. , Schmelzer Robert , Thompson Paul D. , White Michael , Kwon Oh Sung 
Published: May 2022

BackgroundMitochondrial dysfunction may be a key mechanism underlying SAMS. Near infrared spectroscopy (NIRS) is a non‐invasive technique of assessing tissue oxygenation and mitochondrial function and has never been evaluated as a diagnostic tool for SAMS.Methods and ResultsSAMS was confirmed using an 8‐week randomized, double‐blind crossover trial of simvastatin 20 mg / day and placebo separated by a 4‐week washout phase in 39 adults with self‐reported SAMS (Age: 65 ± 6 y; 25 female, 14 male). Tissue oxygenation was measured using NIRS during a handgrip exercise protocol at increasing intensities of maximal voluntary contraction (MVC) before and after statin or placebo treatment. Patients were considered to have SAMS only if they had muscle symptoms on simvastatin and not placebo. Similar to our previous trials, 17/39 (44%) patients were confirmed as having SAMS. There was a significant interaction (p < 0.01) between placebo vs. statin, pre‐ vs. post‐treatment, and SAMS/not SAMS. Tissue oxygenation for average (20‐40%) MVC was significantly lower with placebo when symptoms were present compared with when participants did not have symptoms on a statin (15.66% ± 28.21% vs. 2.35% ± 19.13%, respectively) (p = 0.02). Neither group changed tissue oxygenation with statin therapy (both p > 0.22; SAMS: 28.0 ± 17.6% vs. 22.6 ± 11.9%; No SAMS: 30.5 ± 18.8% vs. 31.0 ± 16.4%, respectively), but the decrease was numerically greater in SAMS patients. There were no significant differences in MVC change score based on symptoms however, patients with SAMS experienced more variable MVC change score responses compared with patients who did not have symptoms.ConclusionsThese results indicate that NIRS may be effective for differentiating patients with SAMS from those with non‐specific muscle pain, however this must be verified with a larger sample.
Sexually Dimorphic Effects of Nociceptor LKB1 on Mitochondrial Metabolism and Mechanical Allodynia After Fasting
Authors: Garner Katherine M. , Burton Michael 
Published: May 2022

BackgroundPain induces metabolic stress in pain sensing neurons (nociceptors); alleviating this stress represents an avenue for treating pain. Liver kinase B1 (LKB1) is involved in maintaining energy homeostasis and is a key mediator of the cellular response to metabolic stress. If cellular energy modulation and pain relief is LKB1‐dependent, then its removal could lead to metabolic imbalance and innocuous stimuli becoming painful (allodynia).HypothesisIn a novel mouse transgenic model where LKB1 is removed from Nav1.8+ nociceptors (Nav1.8LKB1‐), we predict nociceptors will have reduced abilities to cope with metabolic stress, leading to energy imbalance and subsequent allodynia.MethodsAfter baselining for mechanical allodynia, animals were fasted for 24hr to induce metabolic stress. A subset of animals had their primary dorsal root ganglia nociceptors cultured for 6 hours for the Seahorse MitoStress assay. The remainder of animals were tested for mechanical allodynia once a day for 5 days after food was returned.ResultsAs expected, a 24hr fast induced mechanical allodynia in a genotype dependent manner, with Nav1.8LKB1‐ animals responding to the fast in a sexually dimorphic manner. While fasting initially caused allodynia in males of both genotypes, Nav1.8LKB1‐ animals took longer to recover. However, in females, only Nav1.8LKB1‐animals exhibited allodynia after fasting. A sexually dimorphic and genotype dependent effect was also seen in neuronal mitochondrial function. In control females, fasting increased neuronal oxidative metabolism, suggesting the LKB1 pathway is necessary to mitigate fasting‐induced energy depletion. Neither fasting nor genotype changed neuronal mitochondrial function in males, suggesting an alternate mechanism was responsible for their initial allodynia.ConclusionsAlthough Nav1.8LKB1‐ animals of both sexes developed genotype‐dependent allodynia in response to a metabolic stressor (24hr fast), only females exhibited a difference in mitochondrial function in response to this fast. These data support existing literature that suggests neuronal processes are responsible for the development of allodynia in females but not males and suggests that these differences are metabolic in nature. However, additional studies are needed to determine how other sex‐ and cell‐specific metabolic processes can contribute to pain development. This includes how the metabolism of immune cells, such as microglia and macrophages, which have been shown contribute to pain in males but not females, can be affected in an LKB1‐dependent manner to reduce a painful phenotype. Acknowledgements: We would like to thank Luz Barron and Han Jeong for their contributions.
Single Molecule Analysis Reveals Dimer Formation of the Norepinephrine Transporter at the Plasma Membrane of Live Cells
Authors: Luethi Dino , Schütz Gerhard J. , Sitte Harald H. 
Published: May 2022

BackgroundMonoamine transporters are the principal regulators of extracellular monoamine levels in the brain. At the plasma membrane, monoamine transporters form oligomers, which may play an important role in transporter trafficking, drug‐induced monoamine efflux, and drug tolerance development. Using single molecule microscopy, we previously reported that the serotonin transporter (SERT) co‐exists as monomers, dimers, and higher‐order oligomers, while the dopamine transporter (DAT) forms monomers and dimers only. Furthermore, whereas SERT oligomers are kinetically trapped at the plasma membrane by the phospholipid phosphatidylinositol (4,5)‐bisphosphate (PIP2), DAT dimerization is PIP2 independent. The subunit stoichiometry of the norepinephrine transporter (NET) is currently unknown. Based on a high degree of sequence homology between DAT and NET, we aimed to test the hypothesis that NET oligomerization resembles the one of DAT.MethodsWe fused monomeric green fluorescent protein (mGFP) to the N‐terminus of human NET and expressed the construct in Chinese hamster ovary cells. We assessed NET oligomerization using single molecule total internal reflection fluorescence (TIRF) microscopy in live cells. In order to determine the role of cell membrane lipids in NET oligomerization, we pharmacologically depleted the cell membrane of cholesterol and PIP2 before imaging.ResultsSingle molecule brightness analysis revealed that NET co‐exists as monomers (~60%) and dimers (~40%) at the plasma membrane. NET oligomerization displayed signs of transporter density dependence and subunit exchange. Cholesterol depletion did not affect NET dimerization, whereas PIP2 depletion resulted in a small but significant decrease in the average oligomeric state (Student's t‐test, P<0.05).ConclusionNET forms monomers and dimers but no higher‐order oligomers, thereby resembling the oligomerization of DAT. However, unlike DAT, NET dimers exchange subunits over time and dimerization is affected by the transporter density and PIP2 concentrations. This study therefore demonstrates that the oligomerization behavior of each monoamine transporters has unique features.
Gene Expression Changes Associated with Acute Mountain Sickness After Rapid Altitude Exposure at The South Pole
Authors: Byrd M. T. , Wheatley‐Guy Courtney M. , Grill Diane E. , Shea Meredith G. , Herman Nicole M. , Johnson Bruce D. 
Published: May 2022

Many individuals travel to high altitude each year. Acclimatization to altitude occurs over the course of several days as the body adequately adapts to function in an environment with reduced partial pressure of oxygen. However, with a faster accent to attitude, comes an increased potential for maladaptations to occur, leading to the development of acute mountain sickness (AMS). Each year scientists and support workers are transported by plane from McMurdo Station in Antarctica (sea level) to the Amundsen‐Scott South Pole Station (2835m). This uniform and rapid deployment to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression, which may help to illustrate the body’s ability to acclimatize to these environmental conditions.PurposeTo detect pathway specific gene expression changes associated with the development of AMS and help identify potential targets that may aid in the prediction of risk in the development of AMS symptoms due to rapid exposure to high altitude.MethodsVenous blood samples were collected from 53 (height 176.65±8.91cm, weight: 80.49±14.36kg) healthy subjects (38 (27 males; 11 females) that did not develop AMS and 15 (10 males; 5 females) that did develop AMS, as defined by the Lake Louis symptom questionnaire, collected at 2 different time points, with the first being at sea level and the second being after 48hr of altitude exposure). Microarray analysis was performed on the peripheral blood mononuclear cells (PBMCs) from the collected samples, and a logistic regression was performed to determine probe set association with AMS.ResultsThere was a total of 178 significant (p<0.05) probe sets, with 156 of the probe sets being associated with AMS and 22 of the probe sets being associated with no AMS. The probe sets were entered into the Reactome Pathway Database (reactome.org). Pathways identified in association with AMS involved immune system pathways (interleukin signaling) and cellular responses to stimuli pathways (oxidative stress induced senescence signaling), and gene transcription pathways (mitochondrial biogenesis signaling, differentiation of hematopoietic stem cells). Interestingly, pathways identified in association with no development of AMS involved cell cycling pathways (mitotic signaling), chromatin organization pathways (chromatin modifying enzymes), metabolism pathways (utilization of ketone bodies) and the circadian clock pathway (circadian gene expression signaling).ConclusionThese findings indicate potential maladaptive responses within the immune system and mitochondrial function, may play a key role in why some individuals develop AMS symptoms. An individual’s ability to sufficiently utilize different metabolic fuel sources and adapt their circadian rhythm may also prevent the development of AMS symptoms.
Statins stimulate voltage‐gated Ca2+ channels in arterial smooth muscle cells to induce rapid vasoconstriction in resistance‐size cerebral arteries
Authors: Zerin Farzana , Menon Sreelakshmi , Hasan Raquibul 
Published: May 2022

Statins are cholesterol‐lowering drugs with a range of beneficial vascular effects. Vascular effects are believed to result from long‐term statin therapy and reduction of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate‐dependent Rho/ROCK signaling. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. Here, we examined acute vascular effects of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms. We used pressurized arterial myography, simultaneous measurement of vessel Ca2+ fluorescence and diameter, pharmacological modulation, RNAi, Western blotting and biotinylation. Intravascular pressure in the mounted cerebral artery segments was gradually increased to 60 mmHg to stimulate the development of myogenic tone. At 60 mmHg, cerebral arteries developed ~36% myogenic tone, after which a cumulative dose response (0.01‐1000 nM) to rosuvastatin was performed and diameter changes recorded. Our data shows that, at 0.1, 1 and 10 nM concentrations, rosuvastatin induced a potent vasoconstriction – an observation never reported previously with any statin. Intriguingly, rosuvastatin, at 100 nM and 1000 nM concentrations (supratherapeutic), abolished myogenic tone and caused robust vasodilation. Rosuvastatin‐induced vasoconstriction reached a plateau at 1 nM concentration, which was used as reference for mechanistic studies. Within 2‐3 minutes of application, rosuvastatin and simvastatin constricted cerebral arteries by ~26 μm and ~24 μm, respectively. Endothelium denudation (intact ~23 μm vs denuded ~25 μm), or mevalonate supplementation did not alter statin‐induced vasoconstriction (control ~28 μm vs mevalonate ~29 μm), suggesting endothelium‐ and HMG‐CoA reductase‐independent mechanism. In contrast, removal of extracellular Ca2+with EGTA (~6 μm) or the application of nimodipine (~5 μm), a selective blocker of smooth muscle cell voltage‐gated Ca2+ channel, CaV1.2, or siRNA knockdown of CaV1.2 each abolished cerebral artery vasoconstriction and reduced [Ca2+]i, indicating that the Ca2+ entry through CaV1.2 plays a critical role here. Application of thapsigargin, a blocker of SR/ER membrane Ca2+‐ATPase pump, SERCA, but not ryanodine, a blocker of ryanodine receptor‐mediated Ca2+ release, fully reversed statin‐induced cerebral artery constriction and reduced [Ca2+]i. Arterial biotinylation data showed that short‐term statin exposure did not alter the surface expression, distribution and function of CaV1.2 channels.Altogether, our data demonstrate that statins at therapeutic concentrations directly stimulate smooth muscle cell CaV1.2 and Ca2+ influx to induce rapid vasoconstriction in resistance cerebral arteries.
Targeting HDAC6 in the Dorsal Root Ganglia Attenuates Peripheral Nerve Injury‐induced Hypersensitivity
Authors: Giosan Ilinca M. , Zimering Jeffery , Serafini Randal A. , Pryce Kerri D. , Zachariou Venetia 
Published: May 2022

Histone deacetylase 6 (HDAC6) is a Class IIb histone deacetylase, which is primarily localized to the cytoplasm and plays an important role in cell structure and dynamism, transcriptional repression, exocytosis and endocytocis. Preclinical evidence has suggested that HDAC6 inhibitors alleviate signs of chemotherapy‐induced peripheral neuropathy (CIPN), such as mechanical allodynia. However, no group to our knowledge has investigated the mechanism of action of HDAC6 inhibitors in a severe nerve injury model, which has a different pathophysiology than milder models such as CIPN. In this study we use genetically modified mice and the tibial spared nerve injury (SNI) model to determine the impact of dorsal root ganglion (DRG) specific HDAC6 knockout in the induction and maintenance of sensory hypersensitivity. Downregulation of HDAC6 in the DRG was achieved by injection of the left sciatic nerve of adult male HDAC6fl\fl mice with AAV8‐CMV‐Cre‐EGFP or AAV8‐CMV‐EGFP vectors. DRG‐knockdown of HDAC6 prevented the development of mechanical allodynia after SNI. Using in situ hybridization (RNAScope), we also demonstrate that HDAC6 is upregulated at three weeks after the induction of nerve injury in L3‐6 DRG neurons. Furthermore, treatment with the peripherally acting HDAC6 inhibitor ACY1215 (Regenacy Pharmaceuticals, Waltham, MA) after SNI leads to full recovery from mechanical allodynia. We are continuing this work by performing bulk RNA sequencing on L3‐6 DRGs from mice who received 21 days of treatment with ACY1215 or vehicle starting at three weeks after SNI or Sham operation, in order to understand the transcriptomic events and upstream pathways associated with recovery from prolonged neuropathic states.Our findings highlight a promising therapeutic role of HDAC6 inhibitors for the prevention or recovery from sensory hypersensitivity behaviors associated with prolonged peripheral nerve injury. Our future work aims to define the mechanisms underlying HDAC6 action in the DRG and identify new pathways associated with recovery from sensory hypersensitivity to influence novel treatment strategies for pain.
Investigating the temperature‐dependent and independent neuroprotective effects of cyclohexyladenosine in SH‐SY5Y neurons undergoing oxygen glucose deprivation
Authors: Huang Yi‐Ge , Holloway Paul , Buchan Alastair 
Published: May 2022

IntroductionStroke is a global leading cause of adult disability and death. Hypothermia is the most potent neuroprotectant preclinically. It acts broadly upon the pathophysiological mechanisms of ischemia‐induced injury. Clinical trials using physical cooling, which triggers compensatory thermogenesis, have failed. Activation of hypothalamic A1 adenosine receptors (A1AR) by N6‐cyclohexyladenosine (CHA) suppresses thermogenesis in several species. Activation of non‐hypothalamic CNS A1AR at 37 °C also reduces neuronal death. CHA has neuroprotective potential, but the contributions of temperature‐dependent and independent effects are unknown. Therefore, we studied the effects of hypothermia and/or CHA upon the viability of neuronal cultures undergoing oxygen glucose deprivation (OGD). Hypothesis Both temperature‐dependent and independent effects of CHA contribute to neuroprotection in SH‐SY5Y neuronal cultures against OGD.MethodsNeuronal OGD model: SH‐SY5Y human neuroblastoma cells were cultured in Dulbecco modified Eagle's medium with 10% fetal bovine serum, penicillin and streptomycin, and maintained at 37 °C in 95% N2, 5% CO2 in a humidified incubator. Cultures underwent 8 h OGD in a hypoxic chamber at 37 or 33 °C, then 24 h of normoxia in standard culture media, to simulate reperfusion. Drug: N6‐cyclohexyladenosine was dissolved in ethanol to a 1 mM stock and used at 1 μM. Treatment groups: Stable cultures were randomly assigned to: normoxia, 8 h OGD at 37 °C and 8 h OGD and 33 °C. Within each group, wells were assigned to CHA 1 μM, vehicle, or medium only (given 24 h before OGD, during OGD and for 24 h after OGD). One‐way ANOVA was used for statistical comparisons. Outcome measures: Cell death was assessed by flow cytometry (Cytek Aurora 4L). Briefly, neurons were detached using EDTA, washed twice, and resuspended to 106 cells per sample. Cells were incubated in the dark with Zombie Red (Biolegend; 15 min, room temperature, 1:1500 dilution) and then Caspase 3/7 (CellEvent; 30 min, 37 °C,1:500 dilution) and washed twice. Samples (>10,000 events) were gated to exclude debris and for subpopulations of healthy/apoptotic/necrotic cells.Results8 h OGD significantly decreased neuronal viability (37.1 ± 0.7%; mean ± SEM) when compared with normoxic control (70.1 ± 4.0%) (Fig. 1). 33 °C hypothermia was significantly protective (51.9 ± 2.9% healthy cells; p<0.05) of neurons. CHA was significantly protective both at 37 °C (46.7 ± 2.8%; p<0.05) and when combined with hypothermia at 33 °C (69.3 ± 2.6%; p<0.05). There were no significant changes in the percentages of early apoptotic or late apoptotic/necrotic cells.ConclusionCHA provides neuroprotective effect that is additive to that provided by hypothermia in SH‐SY5Y cells. Therefore, it is likely that neuroprotection from CHA in vivo not only can be achieved via its hypothermic effects but also direct temperature‐independent effects on presynaptic cortical neurons.
In Vitro FRET‐Based Helicase Activity Assay as a Drug Screening Tool
Authors: Sheehan Hugo , Fernander Gaddiel , Nyman Karl , Borysov Sergiy I. 
Published: May 2022

The Cdc45‐Mcm2‐7‐GINS (CMG) helicase is a protein complex that unwinds double‐stranded DNA into single strands during DNA replication at the S‐phase. Previous research has pointed to the CMG helicase as a potential target for cancer treatment. Indeed, the CMG helicase activity is an essential and vulnerable component of the cell cycle machinery of cancerous cells. However, the development of anti‐cancer helicase targeting molecules requires a robust and quantifiable helicase assay.Here we report a novel CMG helicase activity assay that utilizes FAM‐BlackFQ dual‐labeled DNA oligonucleotides as a substrate. The underlying principle of the Fluorescence Resonance Energy Transfer (FRET) allows for a quick, reliable, real‐time measurement of helicase activity in vitro. As a source of active CMG helicases, we prepared a nucleoplasmic extract (NPE) derived from the cell‐free system of Xenopus egg extracts, a canonical system widely used to study vertebrate DNA replication in vitro. This NPE is a rich source of the Xenopus CMG helicase, which is highly analogous to its human counterpart.In this report, we present optimization of our novel assay. Remediation of potential contaminations with endogenous DNases is addressed. We utilized our helicase assay to study inhibition of the CMG helicase by therapeutically relevant peptides. We also extended its utility by screening natural compounds of the NCI/DTP library N5.
Prenatal Hypoxia is Associated with Coronary Artery Endothelial Dysfunction in Male and Female Offspring
Authors: Hula Nataliia , Pasha Mazhar , Quon Anita , Kirschenman Raven , Cooke Christy‐Lynn M. , Spaans Floor , Davidge Sandra T. 
Published: May 2022

IntroductionFetal hypoxia is a common consequence of complicated pregnancies and is linked to the development of cardiovascular (CV) dysfunction in the offspring. We showed an impaired cardiac function in adult prenatal hypoxia offspring, however, the mechanisms are not known. Coronary artery endothelial dysfunction has been shown to contribute to the development of cardiac dysfunction, however, whether endothelial function is impaired in coronary arteries of prenatal hypoxia offspring is unknown. We hypothesize that prenatal hypoxia leads to impaired coronary artery endothelial function in adult male and female offspring.MethodsPregnant Sprague‐Dawley rats were exposed to normoxia (21% O2) or hypoxia (11% O2; p‐Hyp) on gestational days 15‐21 (term=22 days). Male and female offspring were aged to 9‐9.5 months. Left anterior descending coronary arteries were isolated (n=5‐7/group), and endothelium‐dependent vasodilation to methylcholine (MCh) and endothelium‐independent vasodilation to the nitric oxide (NO) donor sodium nitroprusside (SNP) were assessed by wire myography. To assess the contribution of endothelial vasodilation pathways, MCh‐curves were performed in the presence/absence of inhibitors of nitric oxide synthase (L‐NAME), prostaglandin H synthase ([PGHS]; meclofenamate), or small‐ and intermediate‐conductance Ca2+‐activated potassium channels (apamin and TRAM‐34, respectively). Data were summarized as Emax (maximum vasodilation response), pEC50 (concentration required to produce 50% of Emax) or Area Under the Curve (AUC). Data were compared by two‐way ANOVA (Sidak's post hoc test); p<0.05 was significant.ResultsMch‐induced vasodilation (Emax) was decreased in p‐Hyp males (p=0.006) and females (p=0.032) compared to normoxia offspring. L‐NAME completely inhibited vasodilation (AUC) in normoxia and p‐Hyp males (p<0.0001). In the female offspring, incubation with L‐NAME completely inhibited vasodilation in the normoxia group (p<0.0001), but to a lesser extent (Emax ~15%) in the p‐Hyp offspring (p<0.0001). In contrast, meclofenamate did not alter MCh‐induced vasodilation (AUC) in males, while in females, meclofenamate increased vasodilation to MCh in the p‐Hyp group only (p=0.04). Apamin and TRAM‐34 did not alter responses to MCh in any of the groups. SNP sensitivity (pEC50) was decreased in p‐Hyp females compared to normoxia controls (p=0.012), without changes in males.ConclusionsOur data suggest that prenatal hypoxia leads to coronary artery endothelial dysfunction in male and female offspring. In prenatal hypoxia males, NO contribution to endothelium‐mediated vasodilation was reduced. In females, prenatal hypoxia increased PGHS‐dependent vasoconstriction and decreased smooth muscle sensitivity to NO. Understanding the mechanistic pathways involved in programming of CV disease may allow for the development of future prenatal and postnatal therapeutic interventions.