bluuebunny/crossref_metadata_2025 huggingface dataset:
Does PVN Neuropeptide Y contribute to the sympathoexcitatory effect of insulin in the arcuate nucleus?
Authors: Cassaglia Priscila A. , Brooks Virginia L.
Published: June 2021

Insulin acts in the Arcuate Nucleus (ArcN) to increase lumbar sympathetic nerve activity (LSNA) and LSNA baroreflex gain (BRG) via a pathway that includes the hypothalamic paraventricular nucleus (PVN). Two ArcN neuronal types project to PVN: melanocortin (MC) and Neuropeptide Y (NPY). Recent data implicates MC in the sympathoexcitatory effect of insulin, but whether NPY also contributes is unknown. To test this hypothesis, we determined if inhibition of PVN NPY type 1 receptors (Y1x) reduces the effect of ArcN insulin to increase LSNA and LSNA BRG. In chloralose‐anesthetized female rats, bilateral microinjection (60nL) of insulin in ArcN (n=3; 0.06nU) increased (P<0.05) LSNA (132±7%) and LSNA BRG (3.9±0.4 to 6.9±0.6 %/mmHg). PVN Y1x (n=5; BIBO3304; 60pmol) also increased (P<0.05) LSNA (161±14%) and LSNA BRG (3.5±0.5 to 5.4±0.9 %/mmHg), indicating endogenous NPY inhibits LSNA. ArcN insulin and PVN Y1x given together (n=5) did not increase LSNA (155±12%; P<0.05) or LSNA BRG (3.4±0.5 to 6.0±1.2 %/mmHg; P<0.05) more than either alone, suggesting a converging pathway. Conversely, microinjection of NPY in PVN (n=3; 6pmol) decreased (P<0.05) LSNA (68±14%) and LSNA BRG (5.8±0.6 to 3.4±0.4 %/mmHg), and PVN NPY and ArcN insulin together (n=3) produced similar decreases in LSNA (63±15%; P<0.05) and LSNA BRG (4.9±1.3 to 2.9±0.9 %/mmHg; P<0.05). Thus, PVN NPY blocked the effect of ArcN insulin. These data support the hypothesis that ArcN insulin increases LSNA and LSNA BRG in part via inhibition of PVN‐projecting NPY neurons. Support: HL088552.
Central melatonin modulates the ventilatory response to hypercapnia but not to hypoxia in anaesthetized rats
Authors: Sanchez Adriana Paula , Delchiaro Adriele , Giácomo João Francisco G , Pupin Juliana P , Luzzi Mayara C
Published: June 2021

In the present study we assessed the participation of melatonin on ventilatory responses to hypercapnia and to hypoxia. To this end the pulmonary ventilation and body temperature of male Wistar rats was measured after microinjecting melatonin (0,5 mg/mL) or vehicle (DMSO) into the fourth ventricle followed by 60 min of hypercapnia (7% CO2) or hypoxia exposure (7% O2). Compared to the vehicle group, the ventilatory response to hypercapnia increased significantly (about 40%) in animals injected with melatonin by elevating respiratory frequency, however no effect was observed on ventilatory response to hypoxia. No difference in body temperature between groups was observed during hypercapnia or hypoxia. These data suggest that melatonin exerts an excitatory modulation on hyperventilation induced by hypercapnia but play no role on hypoxia‐induced hyperventilation.

Financial Support: FAPESP, FIPA.
Intermittent Hypoxia Alters Circulating Leptin Levels and the Activity of Pro‐opiomelanocortin (POMC) Hypothalamic Arcuate Nucleus Neurons
Authors: Moreau Jason Michael , Messenger Scott Allan , Ciriello John
Published: June 2021

Intermittent hypoxia (IH) has been shown to induce a reduction in body weight; however, the mechanisms associated with this affect are not known. In this study, adult male Sprague‐Dawley rats were exposed to eucapnic, isobaric IH (80 s hypoxia to 6.5% oxygen; 120 s normoxia) or similarly to normoxic conditions for an 8 h period during the rat sleep cycle. Immediately after exposure, a blood sample was collected for determination of plasma leptin, and the animals were then perfused transcardially for immunocytochemistry. Brains were removed, paraffin embedded, and transverse sections of the hypothalamus were cut at 6 μm and processed for the immediate early gene Fra‐1 and POMC expression. In second set of animals, food intake was measured for 14 h immediately after IH exposure. The animals exposed to IH had a significantly greater decrease in body weight compared to normoxic controls, and consumed significantly less food despite their loss in body weight. Also, IH animals had a significantly elevated plasma leptin level compared to normoxic controls. Finally, an increase in Fra‐1 expression in POMC arcuate neurons was found within the IH exposed animals. These data suggest that the increased plasma leptin induced by IH may alter the activity of arcuate POMC neurons that in turn alter central mechanisms regulating energy balance. Supported by Heart and Stroke Fdn. of Ontario.
Oxidant stress, inflammatory and autonomic pathways in the development of atrial fibrillation
Authors: Van Wagoner David R
Published: June 2021

Atrial fibrillation (AF) affects more than 3 million Americans. AF promotes heart failure (HF) and is associated with increased risk of stroke and mortality. While AF promotes electrophysiologic changes in atrial myocytes and changes in the pathways of electrical activation, ion channel blockers have proved largely ineffective in treating or preventing AF. To identify the causes of AF, recent studies have focused on “upstream” pathways that precede its onset. Advanced age is the strongest risk factor for AF, but male gender, hypertension, valvular heart disease and HF also increase risk of AF. Hemodynamic stress increases atrial endothelin‐1 abundance, contributing to NADPH oxidase (NOX) expression and activity. NOX activity contributes to atrial electrophysiologic and structural remodeling that promotes persistent AF. Autonomic activation secondary to heart failure contributes to dysregulation of intracellular calcium handling and ectopic electrical activity. Both oxidant stress and autonomic activation promote systemic inflammation and inflammatory cell activation. Statins have been shown to have some antioxidant and anti‐inflammatory activity, but their impact on AF is limited. It remains to be determined whether other therapies that more specifically address upstream signaling pathways can help to prevent AF or improve the management of this common but vexing aging‐related condition.
Tissue tension supports the in vitro co‐culture of ras‐keratinocytes and myofibroblasts
Authors: Barua Prognanwita , Lam Christina C. , Vaughan Melville B.
Published: June 2021

Tissue tension is necessary to support the myofibroblast, a cell derived from a fibroblast and present in wound healing and tumor stromas. Currently‐used skin equivalent models lack the ability to generate tension. Our goal was to determine whether a tension‐maintaining skin equivalent model supports myofibroblast growth and the growth of ras‐keratinocytes, precancerous cells previously shown to modulate their phenotype in response to their environment. We developed a dermal equivalent with a plastic ring within a liquid collagen/fibroblast mixture and converted it to a skin equivalent by overlaying with keratinocytes. Skin equivalents were cultured at the air/liquid interface. Replicate tissues were harvested weekly and prepared for immunofluorescence staining. Myofibroblasts in the dermis were identified by alpha‐sm actin staining in stress fibers; ras‐keratinocytes differentiated and stratified atop the dermal compartment. Proliferation of both cell types was identified by EdU incorporation and staining. Interestingly, the keratinocyte strata consisted of disorganized clumps of cells rather than organized layers even though invasion into the dermal compartment was inhibited. The tension‐maintaining skin equivalent supports ras‐keratinocytes and can be used to study the role of myofibroblasts during cancer progression in vitro.

Grant Funding Source : The University of Central Oklahoma and Grant Number P20RR016478 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH).
Growth change in cartilaginous Eustachian tube angulation and its relationship with age at onset of otitis media
Authors: Pagano Anthony Santino , Wang Eugene , Laitman Jeffrey T.
Published: June 2021

Middle ear infection (otitis media, OM) is amongst the most common childhood diseases in the United States. Despite this, there is a lack of data on growth of the Carilaginous Eustachian tube (CET) and how this may relate to onset of OM. This study investigated growth changes in reconstructed CET dimensions. Its length (between its orifice on the temporal bone and medial pterygoid tubercle) and orientation (angle made with the medial pterygoid plate posterior edge) were quantified in a dry cranial growth series (n=258) divided into dental eruption stages. 3D landmark coordinate data were digitized from each specimen using a Microscribe digitizer. A total of 54 landmarks on the face, upper respiratory tract, and temporal bone were used. These data underwent Procrustes scaling so that relative dimensions could be directly compared between growth stages with Bonferroni‐adjusted T‐tests. Results showed no significant growth change in relative CET length but CET angulation underwent marked and significant (p<0.05) growth change between infancy and adulthood with greatest amount of change occurring within the first three years of life, coinciding with peak onset age for OM. The results suggest that the development of OM may be related to CET angulation and absolute length.

Grant Funding Source : Mount Sinai School of Medicine
Changes in adult fibroblast populations with passaging reduce reprogramming efficiency
Authors: Su Elaine Jean , Downing Timothy , Li Song
Published: June 2021

Induced pluripotent stem cell (iPSC) technology has immense therapeutic potential to treat injury and a variety of degenerative diseases. Previous research has suggested an elite model for the reprogramming of adult fibroblasts into iPSCs; in this model, a rare existing population of primary cells preferentially reprogram to pluripotency. While it is postulated that this elite population of cells could have adult stem cell origins, few studies have been able to identify the key attributes of such populations. Here, we explore the “reprogrammability” of a new adult stem cell population and study how changes in these cells over time (i.e. higher passages) undergo phenotypic changes that may contribute to decreased reprogramming efficiency. Preliminary results indicate a significantly higher reprogramming efficiency in lower passage cells; these data suggest that population changes and perhaps differentiation of progenitor cells in higher passages result in decreased number of “elite”, reprogrammable cells. This model is important for evaluating reprogramming mechanisms as well as for culture techniques for future clinical applications.

Grant Funding Source: CIRM
Umbilical cord matrix stem cells trophic ability and use as a drug delivery vehicle for treatment of melanomas
Authors: Davis Kelsey M.
Published: June 2021

Melanoma is caused by uncontrolled growth of melanocytes, which can lead to a progressive form of malignant melanoma with limited treatment options. Stem cells have previously demonstrated a trophic ability to melanoma, which could be used as an anti‐cancer delivery system. Interferon‐β (IFN‐β) has been used as an anti‐cancer drug in previous studies. Our aim was to use umbilical cord matrix stem cells (UCMSC) loaded with IFN‐β (UCMSC‐IFN‐β) as drug delivery vehicles for melanoma treatment. To investigate the trophic ability of UCMSC towards B16F10 melanoma cells, we plated B16F10 melanoma cells at increasing concentrations in the bottom of a transwell plate and UCMSC's plated in the top of transwells. After 24 hours, the number of UCMSC's showing trophic ability was counted. All the experiments were replicated three times. To investigate the anti‐cancer ability of UCMSC‐IFN‐β on B16F10 melanoma cells, we plated B16F10 melanoma cells at increasing concentrations in the bottom of a transwell plate. UCMSC and UCMSC‐IFN‐β were then plated in the top of the transwells. After 48 hours, the number of B16F10 melanoma cells both alive and dead was counted. All the experiments were replicated three times. This in vitro study shows that as melanoma cells concentration increased, the amount of UCMSC migration increased as well. The second study showed that wells with UCMSC‐IFN‐β had higher amounts of dead B16F10 melanoma cells as compared to the control wells with UCMSC. These results indicate the trophic ability of UCMSC's and the use as a drug delivery vehicle through the anti‐cancer ability of UCMSC‐IFN‐β.

Grant Funding Source : A.T. Still University Graduate Program
Labeling of Porcine Mesenchymal Stem Cells (MSCs) with MRI contrast agent Ferex for use in Abdominal Aortic Aneurysm (AAA) Models
Authors: Faries Peter , Rawal Bhakti , Saeboe Karen Briley
Published: June 2021

Introduction

Reliable methods of labeling and tracking MSCs are necessary to allow non‐invasive evaluation of their effects on aneurysm. Ferex is a super paramagnetic iron compound that when placed intracellularly, can be identified by magnetic resonance imaging (MRI). This study aims to evaluate Ferex as an in vivo cellular label in Porcine AAA models.

Methods

MSCs were isolated from the pig's bone marrow via ficoll plaque separation and expanded in culture. MSCs were incubated with 100ug/ml conc. of Ferex and a cationic transfecting agent, Poly‐L‐Lysine (PLL). Around 10 million Ferex labeled MSCs were then transplanted to the pig after a surgery that induced AAA. MRI was performed at 3 Tesla three times over a two week time period post surgery at day 4, 7 and 14 respectively. A longitudinal survey scan was performed to locate the AAA within the tissue. Time‐of‐Flight (TOF) and T2 weighted images were obtained to evaluate the vasculature and flow with in the AAA as well as to identify the iron laden cells within the arterial wall. Following the T2‐weighted scan, T2*‐weighted multiple echo gradient echo sequences were applied in order to quantify the signal on a pixel‐by‐pixel basis.

Results

As shown in Figure 1, the iron labeled cells were visible (as signal loss) at all time points post transplant. T2* values strongly indicate that the iron cores remained within the labeled stem cells (and were not exocytosed and/or phagocytosed by other cells). The percent relative change in the normal arterial wall was 0.8 +/− 2%, respectively for all time points tested. Figure 2 summarizes the results obtained from the Perl's’ staining. Strong correlation between the in vivo MR signal and iron deposition (blue color on Perl's) was observed.

Conclusion

The results clearly support the hypothesis and show that it is possible to detect iron over a two week time interval post transplant accompanied by strong correlation between MR and histology.
RU486 improves cutaneous wound healing in mice submitted to psychological stress
Authors: Monte-Alto-Costa Andrea , de Almeida Tais Fontoura
Published: June 2021

Cutaneous wound healing is a complex process involving inflammation, granulation tissue formation, and remodeling, which result in scar formation. Chronic stress impairs cutaneous wound healing through the activation of the hypothalamic–pituitary– adrenal axis (HPA). Among the products of HPA are glucocorticosteroid (GC) hormones, which have been shown to delay wound closure. Therefore, we evaluate the effects of administration of a GC receptor antagonist (RU486) on cutaneous wound healing of chronically stressed mice. Male mice were stressed by spun at 115 rpm for 15 min per hour, daily, until euthanasia. A group of mice were only chronically stressed. A group of chronically stressed mice received daily I.P. injection of RU486 (20mg/Kg). Control mice were not subjected to stress, but received RU486 daily. An excisional lesion was made and measured. Fourteen days later, mice were killed to collect blood and lesions. The inflammatory infiltrate, angiogenesis, mieloperoxidase (MPO), and collagen deposition were evaluated in wounds. Stressed RU486‐treated mice showed a worst rate of contraction and re‐epithelialization than RU486‐treated controls. Stressed RU486‐treated mice demonstrate a recovery of MPO activity when compared to stressed animals. Our preliminary results indicate that blocking GC signalization improve wound healing in stressed mice. Supported by CAPES, FAPERJ & CNPq.
Comparison of in vitro adherence, proliferation and osteogenesis of equine bone marrow‐derived mesenchymal stem cells to identify an allogeneic donor
Authors: Dhar Madhu , Neilsen Nancy , Amelse Lisa , Hurst Sarah , Carter-Arnold Jessica
Published: June 2021

Equine mesenchymal stem cells (eMSCs) are promising candidates to repair and to regenerate damaged tissue in horses. To avoid immune reactions, the use of autologous eMSCs is desired, but the number of cells obtained from biopsies is generally too low for direct implantation. As a result, it takes several weeks for in vitro expansion of eMSC cultures to generate enough numbers for injection into the same horse. The only way to satisfy this high demand for eMSCs is the use of allogeneic eMSCs. eMSCs lack major histocompatibility complex II antigens and are able to avoid immune recognition allowing for allogeneic administration of these cells. Similar to humans, equine bone marrow‐derived MSCs exhibit variability in their rates of proliferation and differentiation in vitro. Experiments were carried out to identify a “reliable” donor and establish MSC cultures in order to generate off‐the‐shelf allogeneic cell stocks for immediate administration into client cases. Results of experiments to compare the cell viability (live/dead staining), proliferation (MTS assay), in vitro differentiation into osteoblasts (alkaline phosphatase and alizarin red staining), and gene expression from 7 horses are presented. Only one out of seven horses demonstrated a high rate of proliferation, and potential for in vitro osteogenesis, hence, was found to be superior to the others. This research is sponsored by a grant from the Center of Excellence in Livestock Diseases and Human Health.
Superfast Electrophoretic Transport of Carbocyanine Dyes In Situ: An Improved Method in Nerve Pathway Tracing
Authors: Heuer Tiffany A , McMaster Brian , Hong Se-Pyo , Henderson Charles
Published: June 2021

Carbocyanine dyes are an important tool used to trace the innervations to specific structures, and although the rate of dye transport has improved, faster rates are required to make these dyes effective especially in fixed tissues. We applied a direct electrical current to the sciatic nerve of a cat to electrophoretically transport pastes of DiI and DiD in situ, using a sodium borate (SB) buffer ‐ filled anode apparatus and hypodermic needle as a cathode. Dye labeling was visualized using a Bio‐Rad Radience 2100MP confocal microscope. DiI was transported at an average rate of 60.2 ± 4.1 mm/hr, which was >5,000 X faster than previously reported for crystal diffusion (Lukas et al. 1998, Swift et al. 2005) and 50 X faster than using a DC field in vitro. The average electrophoretic transport rate of DiD (60.9 ± 7.0 mm/hr) was similar to DiI. Lower crystal solubility and reverse micelle formation may be responsible for the slower diffusion rates reported previously. Use of the paste form with a lower alcohol content together with SB buffer solution, could reduce reverse micelle formation, and improve diffusion. Initial counter‐ion distribution at the anode may facilitate development of superfast electrophoresis in a weak field (5mA). This could allow the small ions to facilitate superfast electrophoretic transport of the carbocyanine dyes at low currents.

Grant Funding Source : Palmer College of Chiropractic
HIF‐1alpha and COX‐2 expression and mouse brain capillary remodeling during adaptation to hypoxia and re‐oxygenation after prolonged moderate hypoxia
Authors: Benderro Girriso , LaManna Joseph Charles
Published: June 2021

Normal brain function is delicately sensitive to continuous and controlled oxygen delivery. One of the ways that the brain maintains its optimal continuous supply of oxygen and nutrients is through brain angiogenic adaptational changes. Hence, the objective of this study was to determine the relative time course of the capillary regression during re‐oxygenation after chronic hypoxic exposure, and expression of some of the signaling factors involved in the process. Four month old male C57BL/6 mice were kept in a hypobaric chamber at 290 torr (0.4 ATM) for 21 days and allowed to recover at normoxia for up to 21 days. The mice were either decapitated or perfused in situ and brain samples collected were either lysated for Western blot analysis or paraffin embedded for immunohistochemistry. Hypoxia inducible factor 1alpha (HIF‐1á) accumulation in hypoxia was observed to be diminished during re‐oxygenation; however, Cyclooxygenase‐2 (COX‐2) expression was elevated during hypoxia as well as re‐oxygenation. Significantly increased capillary density at the end of the 3rd week of hypoxia regressed toward normoxic level as the duration of re‐oxygenation was prolonged. In conclusion, increased COX‐2 expression during hypoxia where angiogenesis occurs and re‐oxygenation, when micro‐vessels regress indicates the involvement of this enzyme in wide range of brain angioplasticity.

Grant Funding Source : NS38632
Embryonic origin of microglial progenitors and their routes of entry into the mouse developing central nervous system
Authors: Chan Wood Yee , Ng Tsz Chung , Chow George W.H. , Rezaie Payam
Published: June 2021

Mircoglia are specialized mononuclear phagocytes within the central nervous system (CNS). Their embryonic origins and routes of entry to the CNS remain controversial. In this study, we used Iba1 as a specific marker for microglial progenitors in mouse embryos and employed multiple immunohistochemical localization and tissue transplantation to establish (1) their routes of entry to the CNS and (2) their origin. Iba1+ microglial progenitors were found in the mesenchyme at E9.5, and started to appear in the neural tube at E10.5. By E13.5, significant numbers of Iba1+ cells were observed in the mantle layer. Examination of sectioned tissues suggested that Iba1+ cells entered the neural tube through its basal surface, apical surface or blood vessels. When fragments of E11.5 liver tissues genetically labeled with eGFP were transplanted to the mesenchyme next to the unlabeled E10.5 neural tube, eGFP+Iba1+ cells were found within the neural tube one day after transplantation, implicating that Iba1+ cells from the embryonic liver were able to migrate through the mesenchyme to enter the neural tube. In conclusion, microglial progenitors expressing Iba1 enter the neural tube through multiple sites, and the embryonic liver could represent a source of origin.

The work was supported by the General Research Fund from the Research Grants Committee of the Hong Kong Special Administrative Region, China (Project No. 461909).
The neuroanatomy of deep brain stimulation for the treatment of Parkinson disease
Authors: Smith Lorinda K. , Dibble Lee , Foreman K. Bo
Published: June 2021

The basal ganglia (BG) contribute to control of automatic movements such as postural reactions. In Parkinson disease (PD), neural degeneration alters the overall output of the BG, causing movement deficits. Deep brain stimulation (DBS) is an established treatment for disabling motor signs in PD that are unmanageable by medication alone. However, DBS has been associated with unwanted side effects including impairment of postural control, which may lead to falls and related injury. The purpose of this narrative literature review was to determine common neuroanatomical locations of DBS implantation and their effect on postural control and falls. This review is a preliminary step towards examination of postural control tasks in patients with DBS implants in order to gain insight into the role of the BG in postural control. The subthalamic nucleus (STN) and the globus pallidus internus are the most common areas of DBS implantation, with STN implants having a higher incidence of adverse postural control effects. DBS implantation in the pedunculopontine nucleus is being studied with mixed results. Understanding of postural control deficits of individuals with DBS for PD may be used to assist rehabilitation professionals in fall prevention efforts and ultimately inform neurosurgeons in placement and programming of DBS to minimize postoperative postural control deficits.

Grant Funding Source: None
Time course of neuronal effects as a result of post‐traumatic stress disorder (PTSD)‐relevant social stress on a mouse model
Authors: Chakraborty Nabarun , Meyerhoff James , Muhie Seid , Hammamieh Rasha , Jett Marti
Published: June 2021

Impaired fear extinction, associated with decreased activation in the ventromedial prefrontal cortex (mPFC), has been reported in subjects with PTSD. In rodents, chronic exposure to stressors induced atrophy in mPFC, associated with disruption of working memory and behavioral flexibility. There is a need to identify neurobiological markers for mPFC dysfunction associated with PTSD. The social stress (SS) model in C57/BL6 mice reliably elicits PTSD‐like behaviors. SS or control (C) mice were housed in a small box (without food or liquid) within a larger cage for 6 h/d for 10 d. SS, but not C mice, were exposed to an aggressor mouse at random intervals (3x/d). Analysis of mPFC in golgi‐impregnated brains showed different responses to SS in multipolar (MP) vs. pyramidal (PRM) cells. 24 h after 10d in SS, MP cells in SS mice had lower dendritic spine density (DSD) and length (SL) with a larger percent stubby spines vs. C, yet were similar to C at 4 wks after 10 d SS. In contrast, PRM cells in SS mice showed no differences from C at 24 h post‐SS. At 4 wks post‐SS, however, DSD and SL increased in C but remained at 24 h in SS, thus, were lower than C at 4wks. Morphologically, at 4wks, the percent mushroom increased and percent stubby decreased in C but not in SS, leaving SS with a higher percent stubby than C. The effect of SS on PRM cells appears to be more persistent over time, suggesting a cell‐specific temporal modulation.
The effects of angiotensin receptor blockade on functional recovery and inflammatory gene expression following spinal cord injury
Authors: Robbins Emily A , Shlifer I. G. , Manning D. , Jones C. B. , Jones T. B.
Published: June 2021

The role of the renin‐angiotensin system (RAS) in inflammation has been documented. However, its effect on neuroinflammation following spinal cord injury (SCI) has not yet been studied. We sought to evaluate the effect of inhibiting RAS on recovery of function and markers of inflammation following SCI. Female, Sprague‐Dawley rats were treated with losartan (angiotensin II type 1 receptor (AT1R) blocker), captopril (angiotensin converting enzyme (ACE) inhibitor) or vehicle (i.p., sid), beginning one day after lateral compression SCI. Functional recovery was assessed using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale. Rats were sacrificed at 7, 14 or 28 days post injury (dpi) and the tissues processed for PCR or immunohistochemical analyses. We show that rats treated with losartan or captopril have improved functional recovery compared to vehicle‐treated rats. Quantitative PCR array revealed the expression of AT1R and AT2R in the spinal cords of injured and uninjured rats and the induction of pro‐inflammatory genes over 28 dpi. AT1R appears to be localized to infiltrating immune cells following SCI. These data indicate that manipulation of RAS by blocking AT1R or ACE improves functional recovery and causes changes in gene expression in rats following SCI. Thus, further exploration of therapeutic intervention of RAS following SCI appears warranted.

Grant Funding Source: Office of Research and Sponsored Programs ‐ Midwestern University
Distribution of Nicastrin in the central nervous system of APP/PS1 double transgenic mice
Authors: Luo Shifang , He Guiqiong
Published: June 2021

Nicastrin (NCT) is an integral component of gamma‐secretase complex that also contains presenilin (PS), Aph‐1 and Pen‐2. gamma‐secretase is involved in the processing of over 60 substrates (eg. amyloid precursor protein, APP). Studies show that NCT is required for substrate recognition and selection of gamma‐secretase. However, little is known about its distribution in central nervous system (CNS) of APP/PS1 transgenic AD mice model and its wildtype controls. We report here, using immunoblotting and immunohistochemical methods, that NCT is widely expressed in all major areas of both AD and control mice, including olfactory bulb, cortex, hppocampus, basal ganglion, thalamus, cerebellum, brainstem and spinal cord. The NCT expression level is elevated in AD mice, and it is distributed much more extensively than circumscribed amyloid deposits. At the early stages of postnatal development, the NCT expression level is relatively high in CNS, but declined gradually in adult mice. At cellular level, NCT is localized predominantly in cell membrane and cytoplasma at early postnatal stages of AD mice, while only distributed at cell membrane in controls. Our results provide an anatomical basis to suggest that NCT may play a multifunctional role for gamma‐secretase in the developing and adult AD mice brain.

This work was supported by National Natural Science Foundation of China (30700885)
Hypoplastic Left Heart Syndrome: Molecular Consequences of Transcription Factor Mutations
Authors: Shay Ashley Elizabeth , Kirwin Susan , Prospero Carol , Pizarro Christian , Funanage Vicky
Published: June 2021

Hypoplastic left heart syndrome (HLHS; OMIM #241550) is characterized by abnormal hypoplasia of the left ventricle and aorta, stenosis or atresia of the aortic arch and mitral valve, an atrial septal defect, and a patent ductus arteriosus. The incidence of this rare disorder is about 1 in every 5,000 live births. These abnormalities lead to a decrease in proper blood flow from the left ventricle to the systemic circulatory system. Failure of the systemic circulation leads to profound cyanosis in patients. Without intervention, this condition is fatal within the first days of life. Studies have indicated that HLHS has a strong genetic basis when compared to other congenital heart defects. We are examining the role of several genes associated with cardiac development by screening exons and partial intronic regions for mutations by direct screening of genomic DNA from HLHS patients. Candidate genes for this study include TBX5, NKX2.5, GATA4, and GJA1 due to the correlation between mutations in these genes and cardiac defects. These results will be compared within a cohort of HLHS patients to determine if there is a genetic basis for their abnormal cardiovascular development. We have identified three mutations in exon 8 of TBX5: Val263Met, Ser276Asn, and Arg279Gln, as well as three variations within intronic regions: c. 148‐19 A/T (Intron 2), c.755‐29 C/T (Intron 7), and c.983‐8 C/T (Intron 8).

Funding: Nemours Research Programs and NIH‐INBRE (Delaware)
Expressional repression of hepatic cytochrome P450 2d in ICR mice infected with Babesia microti
Authors: Shimamoto Yoshinori , Watanabe Kensuke , Sasaki Mizuki , Ikadai Hiromi , Ishizuka Mayumi , Hoshi Fumio , Yokoyama Naoaki , Igarashi Ikuo , Kitamura Hiroshi
Published: June 2021

We previously reported that Babesia microti infection repressed expression and activity of hepatic drug metabolizing enzyme cytochrome P450 (Cyp) 3a in ICR mice (J. Vet. Med. Sci. 2011, in press). The purpose of this study is to investigate effects of B. microti infection on Cyp2d, another major isoform in the mouse liver. Twelve days after the infection, we observed decrease of transcripts for hepatocyte nuclear factor (HNF)‐4, which is responsible for Cyp2d expression. In agreement, Cyp2d9 mRNA and protein were clearly decreased on 12 days. Moreover, bunitrolol metabolism assay indicates Cyp2d activity was also repressed on 12 days. Babesia infection induced mRNAs of hepatic TNF‐α and NOS2, both of which are supposed to regulate Cyp expression in the liver. Collectively, Babesia decreased Cyp2d‐dependent metabolism in mice possibly by activation of the TNF‐α‐NOS2 axis.
Early postnatal striatal gene expression networks in rat: relevance to schizophrenia
Authors: Novak Gabriela , Fan Theresa , George Susan R
Published: June 2021

Abnormal striatal development is thought to play an important role in a number of diseases, including schizophrenia. In humans, stress during the second trimester of pregnancy leads to increased risk for schizoaffective disorders in the offspring. This is period of active development of the striatum and corresponds to the second postnatal week of striatal development in rat. We show that during the first two postnatal weeks in rat an entire gene expression network becomes down‐regulated and replaced by a mature gene expression network. Using subtractive hybridization, we identified 31 additional genes involved in this process, including 12 novel transcripts with strict developmental expression. We show that in an established schizophrenia model in rat, this developmental process is altered and results in overexpression of the dopamine 2 receptor (D2R) during the second postnatal week, with no change in expression of D1R or other genes. This may play an important role in the development of a schizophrenia‐like phenotype, as striatal D2R upregulation has been shown to cause dysregulation of the prefrontal cortex.
ERK2‐dependent activation of c‐Jun is required for nontypeable H. influenzae‐induced Cxcl2 up‐regulation in the inner ear fibrocytes
Authors: Woo Jeong-Im , Oh Sejo , Lim David , Moon Sung
Published: June 2021

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune‐privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of pro‐inflammatory molecules derived from middle ear infection. Since middle ear infection is highly prevalent in children, middle ear infection‐induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable H. influenzae (NTHi), a major otitis media pathogen, and up‐regulate a monocyte‐attracting chemokine through TLR2‐dependent NF‐κB activation. Here, we aimed to determine molecular mechanism involved in NTHi‐induced cochlear infiltration of polymorphonuclear cells (PMNs). The spiral ligament fibrocytes (SLFs) were found to release Cxcl2 in response to NTHi via activation of c‐Jun, not NF‐κB, leading to PMN recruitment to the cochlea. We also demonstrated that MEK1/ERK2 signaling pathway is required for NTHi‐induced Cxcl2 up‐regulation in the SLFs. Two AP‐1 motifs in the 5′‐flanking region of Cxcl2 appeared to function as a NTHi‐responsive element, and the proximal AP‐1 motif was found to have a higher binding affinity to NTHi‐activated c‐Jun than the distal one. Our results will enable us to better understand the molecular pathogenesis of middle ear infection‐induced inner ear inflammation. [Supported in part by NIH grants DC8696, DC5025, and DC 6276]
Estrogen and Vitamin D Control of Transcription in MCF‐7 Cells
Authors: Magill Jessica N. , Schmitt John M.
Published: June 2021

The Extracellular Signal‐Regulated Kinase (ERK) is part of a key, signaling pathway that regulates both transcription and translation in many cell types. Increases in intracellular calcium levels results in the CaM Kinase‐dependent activation of ERK and cell growth in MCF‐7 breast cancer cells. ERK has also been shown to play a role in the regulation of MCF‐7 cell proliferation through control of downstream transcription factors including Elk‐1. The hormone, Vitamin D has been suggested to play an inhibitory role on cancer cells by blocking ERK activation. Our goal was to evaluate the ability of E2 to activate Elk‐1, through a CaM Kinase/ERK dependent pathway, in MCF‐7 cells. We also examined Vitamin D's inhibitory regulation of ERK and Elk‐1 activation. Interestingly, E2 stimulation of MCF‐7 cells triggered Elk‐1 phosphorylation an effect that was blocked by inhibiting either CaM KK or ERK. Similarly, E2 treatment of MCF‐7 cells also triggered a significant increase in Elk‐1‐dependent luciferase activity. siRNA inhibition of CaM KK or ERK blocked E2‐stimulated Elk‐1 luciferase activity. Additionally, E2 triggered a sustained increase in ERK and Elk‐1 phosphorylation, both of which were blocked by Vitamin D treatment. Vitamin D treatment of cells also inhibited Elk‐1 luciferase activity downstream of E2 stimulation. In summary, our data suggests that E2 utilizes both CaM KK and ERK to activate Elk‐1 transcriptional activity an effect that is blocked by the hormone Vitamin D.
Effect of statins on heme‐oxyegnase‐1 in macrophages and NIH 3T3cells
Authors: Habib Aida , Mrad May F. , Mouawad Charbel A , Al-Hariri Moustafa , Alam Jawed
Published: June 2021

Heme‐oxygenase‐1 (HO‐1) is an antioxidant and anti‐inflammatory enzyme responsible for the degradation of heme into biliverdin, carbon monoxide and iron. Statins have pleiotropic effects in addition to lowering cholesterol. We have inverstigated the effect of statins on HO‐1 in Raw 267.4 cells and primary cells. Induction of HO‐1 was obtained with 25 μM simvastatin or 10 μM fluvastatin in both Raw 267.4 and peritoneal macrophages. Statins alone increased NO formation and iNOS expression in Raw 264.7 cells and pretreatment with inhibitors of NO synthase, l‐NMMA and 1400W blocked this effect. This induction was NO independent in primary eMPM cells. Both statins increased the promoter activity of the mouse proximal and distal promoter in Raw 264.7 cells, effect inhibited by mevalonate and 1400W. Gel retardation experiments were performed and both statins induced nuclear protein‐DNA complexes compared to untreated cells after 12 or 24‐hour incubation with probes for C/EBP, which were supershifted with antibodies specific for C/EBP β, or AP‐1 but not USF. In NIH 3T3 fibroblast cells, statins induced a significant increase in HO‐1 protein and mRNA levels and stimulated activity of mouse HO‐1 promoter (−1287 bp to +73 bp)/luciferase reporter gene by 3.25 ± 0.23 (Mean ± S.E.M., n= 15, p<0.001, t‐test) and 3.13 ± 0.33 (Mean ± S.E.M., n= 6, p<0.001, t‐test), respectively. Gel retardation experiments for C/EBP and USF on statin‐treated cells showed significant nuclear protein‐DNA complexes which were supershifted with antibodies specific for C/EBP or USF. In summary, statins induce HO‐1 and involve signaling molecules and mechanisms that vary among different cell types.
Biosynthesis and transport of cell wall glycolipids in mycobacteria
Authors: McConville Malcolm John , Rainczuk Areck , Yamaryo-Botte Yoshiki , Pyke James , Coppel Ross , Crellin Paul
Published: June 2021

The genus Mycobacterium contains a number of medically important bacterial pathogens. The most important of these, M. tuberculosis, infects one third of the world's population and causes more than 2 million deaths each year. The unusual cell wall of mycobacteria contribute to the pathogenesis, host immune responses and inherent resistance of these bacilli to common antibiotics and antiseptics. The mycobacterial wall comprises macromolecular components (peptidoglycan, arabinogalactan and covalently linked mycolic acids) as well as free glycolipids, such as trehalose mycolates, phosphatidylinositol mannosides (PIMs) and lipoarabinomannan (LAM). While progress has been made in identifying enzymes involved in the synthesis of different cell wall components, little is known about the transport of precursors from the inner leaflet of the plasma membrane to the periplasmic space. Using a combination of transposon mutagenesis, genome wide bioinformatics searches and genetic studies in the related bacterium Corynebacterium glutamicum, we have identified genes that are essential for both the assembly and transport of mature PIMs, LAM and trehalose mycolates. These findings point to the presence of multimeric protein complexes that are involved in regulating flux of intermediates and balanced cell wall synthesis.
Regulation of nitric oxide synthesis by beta‐adrenergic receptors in the nuclear envelope
Authors: Vaniotis George , Merlen Clemence , Tadevosyan Artak , Villeneuve Louis , Chatenet David , Zamboni Robert , Fournier Alain , Hebert Terence , Allen Bruce
Published: June 2021

Both β‐adrenergic receptors (β‐ARs) and endothelin receptors (ETRs) have been shown to be present on nuclear membranes in adult cardiac ventricular myocytes. These nuclear‐localized receptors are functional with respect to ligand binding and effector activation. Furthermore, isoproterenol (ISO) increases transcription initiation in isolated nuclei whereas endothelin‐1 (ET‐1) has the opposite effect. Here we investigate whether ISO and ET‐1 regulate nitric oxide (NO) production in nuclear membranes. Isolated nuclei were incubated with a fluorescent NO indicator, 4,5‐diaminofluorescein (DAF‐2), followed by either ET‐1 or ISO treatment. In both cases, an increase in DAF‐2 fluorescence was observed: this increase was inhibited by the NO synthase inhibitor, L‐NAME. Pertussis toxin treatment also increased DAF‐2 fluorescence. The PKB inhibitor triciribine, which converts ISO from an activator of transcription to an inverse agonist, potentiated ISO‐mediated DAF‐2 fluorescence without affecting ET‐1 signalling. These experiments were repeated in intact myocytes using caged analogues of ISO and ET‐1, and in combination with 4,5‐diaminofluorescein diacetate (DAF‐2 DA), a cell‐permeant form of DAF‐2. Our results suggest the nuclear β‐ARs and ETRs regulate NO synthesis.
Activation of transcription of hepatic HMG‐CoA reductase by insulin requires tristetraprolin
Authors: Ness Gene Charles , Edelman Jeffrey
Published: June 2021

As part of a study of the actions of AU‐rich RNA binding proteins, we noted that in vivo electroporation of siRNAs to tristetraprolin (TTP) completed eliminated transcription from wild type HMG‐CoA reductase luciferase construct in normal rats. siRNAs to several other AU‐rich binding proteins had little effect except that two siRNAs to HuR (1 of 4 mammalian homologs of Drosophila embryonic lethal abnormal vision proteins) also decreased transcriptional activity of HMG‐CoA reductase. TTP mRNA is increased within 10 minutes after giving insulin to fibroblasts overexpressing normal human insulin receptors. Since insulin acts within 2 hrs to increase transcription of hepatic HMG‐CoA reductase, we investigated the effect of TTP siRNAs on the insulin stimulation of HMG‐CoA reductase transcription by performing in vivo electroporation experiments. TTP siRNAs eliminated the insulin mediated increase in transcription. In comparison, siRNA to HuR reduced the increase in transcription due to insulin. siRNA to AUH (AU RNA binding protein/enoyl coenzyme A hydratase) had no effect on transcription. TTP is a CCCH tandem zinc finger protein widely known to bind to AU‐rich elements in RNA and cause mRNA degradation. This study demonstrates that TTP also participates in activation of transcription of hepatic HMG‐CoA reductase in vivo. (Supported by research grant R01 DK 075414.)
Extracellular Matrix Modulates Cytokine Gene Expression in Endothelial Cells
Authors: Kovala Tom , Holmes Zachary , Rossi Laura
Published: June 2021

Signalling initiated by vascular endothelial growth factor (VEGF) and sphingosine 1‐phosphate (S1P) induce angiogenesis. In endothelial cells, Janus kinases (JAKs) are activated and stimulate the signal transducers and activators of transcription (STAT) proteins that regulate target gene expression. Inhibition of JAKs blocked both migration and morphogenesis. The expression of 27 soluble cytokines in S1P‐ and VEGF‐treated cells was examined using Bio‐Plex technology and IL‐6, IL‐8 and MCP‐1 were found to be induced with either stimulus. Basal and induced expression was significantly enhanced in cells grown on the extracellular matrix proteins collagen I, fibronectin and laminin I while collagen IV and matrigel had little effect. Pharmacological inhibitors demonstrated that IL‐6 and MCP‐1 expression is dependent on JAK1/2, STAT3 and STAT5 activation, whereas IL‐8 expression was dependent on JAK1/2 and STAT5. NF‐kappaB activation was essential for expression of the cytokines while ERK and Src also played important roles. Phosphoprotein analysis demonstrating NF‐kappaB and STAT activation supported their role in the induction of gene expression. STAT regulated gene expression was confirmed with luciferase based promoter studies. Both VEGF and S1P induce expression of proangiogenic and proinflammatory cytokines through activation of the NF‐kappaB and JAK‐STAT pathways in endothelial cells.
Characterization of a metal‐dependent regulator protein from Thermobifida fusca
Authors: Novak Walter R.P. , Lu Zeyu Mike , Granger Joseph B. , Ferguson Jordan B. , Santa Maria Peter J.
Published: June 2021

Iron dependent regulators are proteins found in a variety of pathogenic bacteria that cause diseases such as tuberculosis and diphtheria, but few such regulators have been thoroughly studied. The purpose of this research is to characterize a metal‐dependent regulator from Thermobifida fusca, the causative agent of farmer's lung. In comparison to homologous regulators, the regulator from T. fusca has a longer N‐terminal helix. Conformational changes have been shown to occur upon metal binding in related proteins, and it has been suggested that this helix is important in DNA‐binding. The target gene that encodes for the T. fusca regulator was cloned into the pET‐28b expression plasmid, and the protein was expressed in BL‐21(DE3) cells. The regulator was crudely purified using a nickel‐affinity column. Gel‐shift assays indicate the protein successfully binds the diphtheria and tuberculosis DNA consensus sequence in a metal‐dependent fashion. This research is supported by the Wabash College Byron K. Trippet and Haines Biochemistry Funds.
Is expression of C4 PPDK and its regulator, the PPDK regulatory protein co‐regulated by light intensity in maize leaves?
Authors: Poudyal Christina , Chastain Chris J
Published: June 2021

Pyruvate phosphate dikinase (PPDK) is a cardinal enzyme of the C4 photosynthetic pathway that catalyzes the regeneration of the primary CO2 acceptor, PEP. In C4 leaves, PPDK activity is regulated to match the level of light incident on the leaf. The bifunctional PPDK regulatory protein (PDRP) catalyzes this up/‐ down regulation by reversible phosphorylation of a PPDK active‐site Thr, with the enzyme inactive in the phosphorylated state. PDRP also functions as the light‐level sensory component of the regulation cycle. To uncover how these two proteins may be co‐regulated at the molecular level in maize leaves we are assessing changes in the transcript and polypeptide abundance during high to low light dynamic (non developmental) acclimation. Initial results indicate that PDRP is a very low abundance mRNA being some eight orders of magnitude lower than the transcript level of its target enzyme. More focused experiments on high light to low light effects on PPDK and PDRP gene transcript level and polypeptide level are currently in progress. The results will be summarized with respect to how molecular expression of the two interrelated C4 pathway genes are coordinated for optimizing photosynthetic response to short‐term changes in light intensity. This research was supported by: US National Science Foundation Grant No. IOS‐ 0642190.