bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metformin Exposure in uteroProgrammes Hypertension in a Sex‐Specific Manner in Adult Offspring of Obese Mice
Authors: Wilsmore Phoebe , Schoonejans Josca , Ashmore Thomas , Blackmore Heather , Fernandez‐Twinn Denise , Giussani Dino , Ozanne Susan 
Published: May 2022

IntroductionMetformin is the first‐line pharmacological treatment for gestational diabetes in many countries. Although effective at improving maternal glycaemia, metformin readily crosses the placenta and could directly affect the fetus. Indeed, metformin exposure in utero increases postnatal adiposity. However, other long‐term effects on offspring exposed to metformin in utero have not been investigated. Here we explored the impact of maternal metformin treatment during obese, glucose‐intolerant pregnancy on offspring cardiovascular function at one year of age using an established mouse model of maternal diet‐induced obesity.MethodsFemale C57Bl/6J mice were fed either a standard chow diet [Control dams (Con)] or an obesogenic diet [Obese (Ob) and Obese‐Metformin (Ob‐Met) dams] from weaning up to and including pregnancy and lactation. Ob‐Met dams received a clinically relevant dose of metformin orally from one week prior to mating until day 19 of gestation. All offspring were weaned onto standard chow fed ad libitum at three weeks of age. At one year of age, systolic blood pressure (SBP) was measured by tail cuff plethysmography in the conscious animal and echocardiography was performed under anaesthesia. Post‐mortem, femoral arteries were isolated and vasomotor reactivity tested using in vitro wire myography. Myography dose‐response curves were analysed using mixed effects models, all other data were analysed by One‐Way ANOVA. Post‐hoc multiple comparisons were performed where appropriate. Significance was accepted when P<0.05.ResultsRelative to controls, female offspring of Ob‐Met dams had increased SBP. However, no differences in SBP were observed in male offspring (Fig.1A). Female offspring of both Ob and Ob‐Met dams had enhanced femoral vasoconstrictor reactivity to K+ (Fig. 1B) but normal cardiac function (Fig. 1C). In contrast, male offspring of both Ob and Ob‐Met dams showed indices of diastolic dysfunction (Fig. 1C) and male offspring of Ob‐Met dams had enhanced femoral vasoconstrictor reactivity to phenylephrine (PE) (Fig.1B).ConclusionsMaternal obesity and metformin exposure in utero caused sex‐specific changes in cardiovascular function in 12‐month‐old offspring. In female offspring, exposure to maternal obesity and metformin programmed hypertension with enhanced peripheral vasoconstrictor reactivity. In male offspring, maternal obesity alone programmed echocardiographic indices of left ventricular restrictive filling which were not corrected by metformin treatment, while combined exposure to maternal obesity and metformin also programmed enhanced peripheral vasoconstrictor reactivity. These findings provide novel insight into the long‐term effects of in utero metformin exposure on the cardiovascular health of the next generation in obese pregnancy, and highlight the importance of considering offspring sex in developmental programming studies.
Acute Intermittent Hypoxia Improves Orthostatic Tolerance in Chronic but not Acute Spinal Cord Injured Rats
Authors: Wainman Liisa , Ahmadian Mehdi , Foster Glen , Ainslie Philip , Rodrigues Perim Raphael , Mitchell Gordon , West Christopher 
Published: May 2022

Spinal cord injury (SCI) causes hypotension and orthostatic intolerance due to disrupted sympathetic input to the heart and vasculature following injury1. A neuro‐therapeutic strategy shown to increase somatic motor function below the injury is acute intermittent hypoxia (AIH). While AIH is known to improve control over respiratory and non‐respiratory motor systems with incomplete cervical SCI2,3, cardio‐autonomic responses to AIH after SCI are unknown. In able‐bodied individuals, AIH increases blood pressure (BP), improves orthostatic tolerance (OT), and heightens sympathetic activity4. In incomplete SCI, intact neural pathways regulating sympathetic nerve activity suggest the potential for AIH to increase sub‐lesional sympathetic activity, thus improving cardiovascular function. Previous work has demonstrated that the response to AIH is greatest in rats at least 4 weeks post‐injury due to reestablishment of serotonergic pathways2, therefore the purpose of this project is to investigate whether AIH improves OT in rats following acute (2 weeks) and/or chronic (4 weeks) high‐thoracic SCI. We believe AIH will improve OT more in chronically versus acutely injured rats.12 Wistar rats underwent T3 300 kdyn SCI. At 14 (n=6) or 28 (n=6) days post‐SCI rats were tracheotomized, ventilated and arterial catheterization was performed to assess BP. Rats were subjected to lower body negative pressure (LBNP; recorded with manometer) to decrease mean arterial pressure (MAP) by 5 and 10 mmHg for 1 min. Rats were exposed to isocapnic AIH (10 x 1‐min FIO2=0.10 separated by 2 min of FIO2=1.00; targeting a PaCO2 of 40 mmHg). LBNP was repeated 90 minutes post‐AIH, the expected peak response. Blood gases were measured pre‐ and post‐AIH. A mixed‐design ANOVA was performed to examine effects of time post‐SCI (group) and AIH (treatment) on change in box pressure required to decrease MAP by ‐5 and ‐10 mmHg.There was a significant group*treatment interaction for the ‐5 mmHg (p=0.011) and ‐10 mmHg LBNP stage (p=0.017). Post hoc comparisons demonstrate that in chronic SCI the box pressure required to drop MAP by ‐5 and ‐10mmHg was increased by 41±16% (p=0.009) and 46±22% (p=0.079) versus pre‐AIH, respectively, implying improved OT (Fig 1).We demonstrate that AIH has the capacity to improve OT in a rodent model of SCI in a chronic, but not acute setting.References1. DeVeau et al. Autonomic Neuroscience(2018)2. Golder & Mitchell Journal of Neuroscience (2005)3. Prosser‐Loose et al. Journal of Neurotrauma (2015)4. Stuckless et al. Journal of Physiology (2020)
Sigmar1 Deficiency Results in Pulmonary Fibrosis Associated with Increased Pulmonary Inflammation and Surfactant Protein Levels in Mice Lung
Authors: Remex Naznin S. , Abdullah Chowdhury S. , Aishwarya Richa , Morshed Mohammed M. , Nitu Sadia , Traylor James , Hartman Brandon , King Judy , Kevil Christopher , Orr Anthony W. , Bhuiyan Md. S. 
Published: May 2022

BackgroundSigmar1 is a ubiquitously expressed multifunctional molecular chaperone protein. Extensive studies demonstrated Sigmar1’s molecular functions in the central nervous system, and mutations in the Sigmar1 gene showed association with different neurodegenerative diseases, including amyotrophic lateral sclerosis and distal hereditary motor neuropathy. Studies also showed the protective roles of Sigmar1 in cardiovascular diseases in animal models of cardiac hypertrophy and heart failure. Recent studies focused on Sigmar1 in COVID‐19 drug repurposing as Sigmar1 was identified to interact with SARS‐Cov‐2 replication proteins using proteomic screening and Sigmar1 ligands showed promising protective effects in clinical trials. Despite all these studies showing a function of Sigmar1 in the respiratory system, the Sigmar1’s expression, localization, molecular and physiological functions in the lung remained unknown.ObjectivesThe main objective of this study was to characterize the expression and localization of Sigmar1 in human and mouse lungs. We also investigated the pathophysiological role of Sigmar1 in the lung using Sigmar1 null mice.ResultsUsing a combination of immunohistochemistry, immunofluorescence, and biochemical experiments, we confirmed the expression of Sigmar1 in rodent and human lungs. Hematoxylin and Eosin staining showed that Sigmar1 global knockout (Sigmar1‐/‐) mice lungs develop altered alveolar structure and organization and higher immune‐cells infiltration compared to Wildtype (Wt) littermate controls. This was associated with an upregulated protein level of phospho‐NFκB in Sigmar1‐/‐ mice lungs. Sirius red and Masson’s trichrome staining of lung tissue sections showed increased collagen deposition, pulmonary fibrotic remodeling associated with elevated fibronectin and phospho‐SMAD2 protein levels in Sigmar1‐/‐ mice lungs. Ultrastructural analysis by transmission electron microscopy (TEM) showed structural alteration in the multilamellar bodies with partial loss of lipid lamellae in alveolar‐type II epithelial cells (AV‐II) in Sigmar1‐/‐ mice lungs. Multilamellar bodies in AV‐II cells serve as secretory organelles releasing pulmonary surfactants into the extracellular space. Elevated levels of surfactant proteins (SP), SP‐A and SP–D, were reported to serve as biomarkers for acute respiratory failure and lung inflammation. Consistent with the TEM data, we also found increased SP (A, B, C, and D) protein levels in the lungs indicating the AV‐II pneumocytes dysfunction in Sigmar1‐/‐ mice.ConclusionOur findings suggested that Sigmar1’s lack of function leads to increased pulmonary inflammation, pulmonary fibrotic remodeling, altered structure and functions of multilamellar bodies, and AV‐II pneumocytes dysfunction in Sigmar1‐/‐mice.
RNA Interference Via shRNA Decreases the Number of Transcripts of Scn8a in Glutamatergic Interneurons in the preBötC
Authors: da Silva Carlos Aparecido , Picardo Maria Cristina D. , Del Negro Christopher A. 
Published: May 2022

Rhythmic neural activity that drives the inspiratory phase of the breathing cycle in mammals emanates from glutamatergic interneurons of the pre‐Bötzinger Complex (preBötC). A longstanding question in neuroscience is: what are the underlying mechanisms for inspiratory rhythmogenesis? One proposed mechanism involves the persistent sodium current (INaP), which is the ionic mechanism that underlies the voltage‐dependent bursting‐pacemaker activity of neurons whose rhythmic burst discharges are putatively obligatory for inspiratory rhythm generation. Specifically in brain tissues, the Scn8a gene codes for Nav1.6 ion channels, and those ion channels give rise to INaP.Different genetic tools can help us to understand the role of specific molecules in the generation of motor behaviors, such as breathing. The short hairpin RNA (shRNA) is an artificial RNA molecule, delivered through adeno‐associated virus (AAV), that can inhibit gene expression and promote long‐term knock‐down of the targeted gene.Here we administered Cre‐dependent shRNA to knock‐down Scn8a (thus Nav1.6) in Cre‐expressing glutamatergic neurons in preBötC and quantified mRNA per glutamatergic neuron. We used adult Vglut2Cre mice (10‐14 weeks old) and injected 50 nL of AAVs carrying the shRNA. We used in situ hybridization (RNAscope® Fluorescent Assay) to label mRNA of Scn8a and to identify transduced glutamatergic neurons in the preBötC for RNA quantification at 2 and 6 weeks after injection. Samples were visualized under the Four‐Channel, Upright Confocal Microscope Body Based on Cerna® System (ThorLabs), and images were acquired using the ThorImage®LS Data Acquisition Software, at 40x magnification. The amount of RNA per cell was quantified using the open‐source software Quantitative Pathology(QuPath). We obtained the mean of the number of transcripts per cell ± the standard deviation (SD), and unpaired t‐test was used to evaluate the statistical difference between the shRNA injected mice and their control group (mice whose viral vector contained a non‐targeting shRNA sequence).Our results show that at 2 weeks, the control group had 27.5±9.5 RNA/neuron, whereas the shRNA‐injected had 12.8±3.5, which represents a 53% of decrease in the number of transcripts (P<0.0001). A similar pattern was observed after 6 weeks of the injection in a different cohort, where control animals showed 35.4±6.1 RNA/cells, and the shRNA group only had 24.1±5.2, representing 32% drop in the transcripts for that group (P<0.0001). This initial protocol allowed us to verify that the gene expression has been knocked down at critical time points, encouraging further studies using shRNA to evaluate whether attenuation of Nav1.6 expression in rhythmogenic preBötC neurons impairs breathing, which could resolve the longstanding question regarding the putatively rhythmogenic role of INaP.
Mice With Monoallelic GNAO1 Loss Exhibit Reduced Inhibitory Synaptic Input to Cerebellar Purkinje Cells
Authors: Feng Huijie , Yuan Yukun , Williams Michael R. , Roy Alex J. , Leipprandt Jeffery , Neubig Richard R. 
Published: May 2022

GNAO1 encodes Gαo, a heterotrimeric G protein alpha subunit in the Gi/o family. Gαo, the most abundant membrane protein in the brain, mediates downstream signaling pathways by itself, or via regulating free Gβγ subunits. To determine the role of Gαo protein in regulating synaptic transmission, we use electrophysiology to study cerebellar molecular layer interneuron ‐ Purkinje cell synaptic transmission in a haploinsufficient loss‐of‐function (LOF) Gnao1 mouse model, manifest with movement abnormalities and enhanced seizure susceptibility. We hypothesize that a LOF Gαo protein will increase GABA release through presynaptic Gαo ‐ coupled receptors.Inhibitory post synaptic currents (IPSCs) and excitatory post synaptic currents (EPSCs) were recorded using standard patch clamp techniques. Spontaneous IPSCs (sIPSCs) were isolated with glutamatergic receptor inhibitors CNQX and AP‐5. Spontaneous EPSCs (sEPSCs) were isolated with the GABAergic receptor inhibitor bicuculline. Miniature PSCs were isolated with the additional perfusion of tetrodotoxin. Cerebellar molecular layer inhibitory neurons were labeled with the Kit‐eGFP method and observed under an inverted epifluorescence microscope. Gαo and Gβ protein expression levels from flash‐frozen mouse brain tissues were determined using Western Blot.Instead of increase, we observe a significant reduction in both sIPSC (40%) and mIPSC (75%) frequency but not amplitude in the Gnao1 mutant mouse model. Both frequency and amplitude of sEPSCs and mEPSCs remain unchanged. On Western blots, levels of Gαo were reduced by about half, as expected, while Gβ levels were unchanged from control. To determine whether this reduction is due to the loss of cerebellar inhibitory interneuron populations, we compare cerebellar interneuron number in the cerebellar molecular layer between Gnao1 mutant mice and their wildtype littermate control. There is a modest reduction (13%) in interneuron number of cerebellar molecular layer in the Gnao1 mutant mouse.The observed reduction in IPSC frequency is not related to Gαo ‐ coupled GABAergic receptors. Also, the modest change in cerebellar interneuron counts does not seem to account for the marked reduction in IPSC frequency. These observations, and the reduced Gαo expression in brain with the normal levels of Gβ, strongly support that a model with a haploinsufficient LOF Gnao1 mutant mainly functions through receptor‐independent regulation of free Gβγ subunits to suppress inhibitory neurotransmitter release in Gnao1 mutant cerebellum.
Mapping Allosteric Sites on Protein Kinases Critical in MAPK Signaling
Authors: Fera Daniela , Lin Sabrina , Naagaard Daniel , Chen Han , Finkelstein Maxwell T. , Glatz Gwendolyn , Hayashi Scout , Hotan Quynn , Lau Benjamin , LeBlanc Julia , Li Kevin , Parker Miller Emma , Ye Ming , Zaidi Hussain 
Published: May 2022

The mitogen‐activated protein kinase (MAPK) pathway plays a critical role in controlling cell cycle progression and cell proliferation. This Ras‐Raf‐MEK‐ERK pathway is constitutively activated in over 80% of melanomas, as well as in some pancreatic, colon, lung, ovarian, and kidney cancers. Anticancer drugs targeting proteins earlier in the pathway, such as B‐Raf and MEK, often lose efficacy due to the development of resistance. Thus, drugs that target allosteric sites hold promise if used in combination with ATP‐competitive inhibitors that have already been developed. We have been working on identifying allosteric sites on key kinases of this pathway that could be used for future drug discovery efforts.We previously found that the alpha‐G helix of B‐Raf is critical for its interaction with MEK and that mutations of several residues along this helix could completely abrogate binding and downstream phosphorylation activity in vitro. With this information, we further analyzed the crystal structure of the B‐Raf‐MEK complex to generate mutations in MEK to determine if the same region of MEK that contacts the B‐Raf alpha‐G helix is important for binding ERK. Additionally, since no complex structure of the MEK‐ERK complex was available, we generated mutations in different regions of the C‐lobe of ERK as a starting point to identify a region of contact. We tested the effects of our mutations on binding using pull downs and biolayer interferometry. We also assessed phosphorylation levels of ERK in vitro.Our MEK mutants displayed no difference in binding to B‐Raf, but did have altered binding to ERK. This suggests that the modes of MEK binding to B‐Raf versus ERK differ. Moreover, our mapping of the ERK interface has led to the identification of a residue on the helix spanning residues 232‐245 that, when mutated, significantly reduces both binding to MEK and phosphorylation of ERK. Additional work is needed to determine if our mutation affects the conformation of a nearby alpha helix of ERK and as a result affects MEK binding or if it is due to a direct effect. Nevertheless, these results demonstrate that MEK‐ERK binding and downstream activity can be altered by targeting sites outside their catalytic region.
Advanced Maternal Age Impairs Uterine Artery Remodelling in the Pregnant Rat
Authors: Wooldridge Amy L. , Pasha Mazhar , Chitrakar Palehswan , Kirschenman Raven , Spaans Floor , Cooke Christy‐Lynn M. , Davidge Sandra T. 
Published: May 2022

Advanced maternal age (AMA, ≥35 years) pregnancies are increasingly common, and are at increased risk for pregnancy complications. We previously showed that ex vivo uterine artery constriction due to increasing intraluminal pressures (myogenic tone) was greater in arteries of AMA vs. young pregnant rats. Aging impairs vascular reactivity and increases vascular stiffness via structural changes (e.g. increased collagen), however, the impact of AMA on adaptations to pregnancy in the uterine artery is unknown. We hypothesized that functional and structural uterine artery adaptations to pregnancy are impaired in aged compared with young rats.Pregnant young (4 months) and aged (9 months; ~35 years in humans) rats were studied on gestational day 20 (term=22 days) with age‐matched non‐pregnant rats. Uterine arteries were isolated and mounted in a pressure myograph. Active tone (calcium present in buffer, n=6‐10) and mechanical properties (circumferential stress and strain; calcium absent, n=10‐24) were assessed. Myogenic tone, circumferential stress and strain were calculated from active and passive curves. Collagen and elastin in the tunica media and intima (combined) were visualized with Masson’s trichrome (collagen) or Verhoeff’s (elastin) stains (n=4‐6). Data are mean area under the curve (AUC, myography) or % positive area (histology) ± SEM; analyzed by two‐way ANOVA with Sidak post‐hoc tests (significance: p<0.05).Between 4‐100 mmHg, myogenic tone did not change with age or pregnancy. However, arteries from young pregnant rats displayed forced vasodilation at higher pressures (100‐160 mmHg) that did not occur in arteries from other groups (p<0.0001). Circumferential stress and strain (Figure 1) increased with pregnancy (p<0.0001) but was significantly lower in arteries from pregnant AMA compared to young pregnant dams (p<0.05). Collagen and elastin (% positive areas) did not change with age or pregnancy overall, however, arteries from young non‐pregnant rats had greater collagen:elastin ratios than those from aged non‐pregnant rats (p=0.021). Arteries from aged non‐pregnant, young pregnant and AMA rats had similar ratios.This study illustrates that aging impacts both structural and functional adaptations of uterine arteries during pregnancy. The lower compliancy of arteries from AMA rats may be due to increased crosslinking of the existing collagen within the artery, as it was not explained by collagen:elastin ratio. These structural differences may contribute to the functional differences, potentially reducing uteroplacental blood supply. Thus, impaired uterine artery adaptations may increase the risk of pregnancy complications in women of advanced age.
Therapeutic implication of SOX9 and YAP1 co‐repression in advanced intrahepatic cholangiocarcinoma
Authors: Hu Shikai , Molina Laura , Tao Junyan , Liu Silvia , Hassan Mohammed , Singh Sucha , Poddar Minakshi , Nejak‐Bowen Kari , Oertel Michael , Raeman Reben , Singhi Aatur , Bell Aaron , Monga Satdarshan P. , Ko Sungjin 
Published: May 2022

IntroductionIntrahepatic cholangiocarcinoma (iCCA) is a liver tumor of increasing incidence and devastating prognosis. The most critical barrier for developing a globally effective therapy for iCCA is molecular heterogeneity caused by diverse molecular drivers and cellular origins in the liver. Clinically, over 90% of iCCAs show alterations of YAP1 and/or SOX9. Therefore, we investigated the therapeutic potential of co‐repression of YAP1 and SOX9 as a wide range therapeutic option for advanced iCCA.MethodsBy immunohistochemistry (IHC) using 2 tissue microarrays, we examined the localization of SOX9 and YAP in clinical iCCA. We also assessed clinical relevance of AKT‐NICD (AN) /YAP (AY)‐dependent hepatocyte‐driven iCCA by bioinformatic analysis comparing their transcriptome with large human datasets. Lastly, we comprehensively investigated therapeutic effect of simultaneous deletion of Sox9 and Yap1 in advanced murine iCCA using respective floxed mice.ResultsOver 90% of human CCA samples (n=108) exhibited nuclear SOX9 and YAP1. We also identified significant upregulation of p‐AKT, SOX9 and YAP1 in HCs of patients with primary sclerosing cholangitis and nonalcoholic steatotis hepatitis, well‐known risk factors for iCCA. While co‐expression of AN led to hepatocyte‐derived lethal iCCA, conditional deletion of either Yap1 or Sox9 dramatically reduced AN‐ iCCA development. Yap1 deletion impaired HC‐to‐biliary epithelial cell (BEC) fate conversion and tumor cell proliferation, while Sox9 elimination only repressed proliferation. Interestingly, following single deletion of either Yap1 or Sox9 we observed a few iCCA tumors expressing either SOX9 or YAP1 but not both. This is also identified in a small subset of human iCCA; SOX9+/YAP1‐ (4.6%) or SOX9‐/YAP1+ (3.7%), suggesting that single deletion of YAP1 or SOX9 is not sufficient to abrogate clinical iCCA. We also found that simultaneous deletion of Yap1 and Sox9 completely prevents iCCA development. Remarkably, co‐deletion of Yap1 and Sox9profoundly decreases fully developed lethal iCCA burden while extending survival. However, singular deletion of either shows no survival benefit compared to control group harboring advanced iCCA as a therapeutic manner.ConclusionsWe elucidate the critical, but distinct roles of Yap1and Sox9 in iCCA development and demonstrate the therapeutic potential of simultaneous targeting both for wide range of iCCA irrespective of drivers. Ongoing work will comprehensively delineate the molecular mechanism how YAP1 and SOX9 compensate each other when either is removed in iCCA, and will be relevant for studying adaptive resistance against the singular targeting of SOX9 or YAP1 in iCCA.