bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential host response to Methicillin‐resistant Staphylococcus aureus and Pseudomonas aeruginosa
Authors: Enkhbaatar Perenlei , Jonkam Collette , Traber Lillian , Connelly Rhykka , Lange Matthias , Hamahata Atsumori , Herndon David , Traber Daniel
Published: June 2021

Despite the outstanding achievement in treatment of sepsis, the mortality rate of septic patients is high. The objective of the study was to describe differential host response to different infectious agents using our well‐established ovine sepsis model.

Method

Sheep were given the injury via tracheostomy under anesthesia. Study groups: 1) sham, (no injury), n=6; 2) Smoke (Smoke inhalation) n=6; 3) PS (smoke inhalation & Ps. aeruginosa (1011CFU) instilled into the lungs, n=6; 4) MRSA (smoke inhalation & MRSA (1011CFU) instilled into the lungs, n=6. After injury, sheep were awakened, maintained on mechanical ventilation, and fluid resuscitated for 24 h.

Results:

Mean arterial pressure was similarly depleted in MRSA and PS groups. The severity of lung injury was more pronounced in PS group, which was associated with higher expression of lung IL‐1, 6, and 8. MRSA group had 2‐fold higher fluid retention associated with significantly higher nitric oxide (NO) production and vascular endothelial growth factor (VEGF). The lung tissue arginase level and IL‐13 was higher in PS group.

Conclusion:

Excess IL‐13 leading to higher arginase activity in PS may result in lesser production of NO. The severe vascular leak in MRSA group may be due to excess NO and VEGF. Differential responses to different pathogenic agents require more focused treatment approach considering the nature of infectious agents and specific host response to it
Nitrosative Stress in an Ovine Model of Acute Lung Injury
Authors: Esechie Aimalohi , Enkbaatar Perenlei , Horvath Eszter , Jonkam Collette , Hamahata Atsumori , Traber Lillian , Traber Daniel
Published: June 2021

AIM: elucidate changes involved in nitrosative stress using an ovine model of ALI.

METHOD: Sheep (n=5 per group) were surgically instrumented for chronic study. 5 days after surgery, the animals were randomly allocated as follows: Injury (instrumented; 3rd degree 40% total body surface area flame burn and insufflation with 48 breaths of cotton smoke under deep anesthesia) and sacrificed at the following time points post‐injury: T=4hr, 8hr, 12hr, and 24hr; control (instrumented; no injury). Data is reported as mean ± SEM. RESULTS: Pulmonary gas exchange decreased 24h post burn and smoke injury (p < 0.05) and correlated with increased lung water content at 18h and 24 h (p< 0.05). Inducible nitric oxide synthase (iNOS) expression increased after injury compared with the control groups; peak iNOS expression was at 12h post burn and smoke inhalation (p < 0.05 vs control). Plasma nitrate/nitrite (NOx) correlated with protein expression: plasma NOx values increased 12h post injury (p = 0.046). 3‐nitrotyrosine (index of reactive nitrogen species) increased after burn and smoke inhalation compared to control (p < 0.05, n=5). DNA damage was assessed by measuring repair enzyme, poly (ADP) ribose polymerase (PARP) which increased in the injury group (p < 0.05 vs control).

CONCLUSION: Therapeutic interventions to mediate excess free radical production could ameliorate impaired pulmonary function associated with ALI.
Hemorrhagic Shock Data Mining Project: Poincare Plot Analysis
Authors: Ward John A. , Cancio Lee C , Batchinsky Andriy I , Newman Paul R. , Convertino Victor A. , Holcomb John B.
Published: June 2021

In this study, Poincare plot analysis predicts volume status in conscious animal models of hemorrhagic shock. We examine beat‐to‐beat systolic pressure variability as a function of volume status. Archived data from 10 conscious sheep that were hemorrhaged from a catheter in the aorta then volume resuscitated are examined. Hemorrhage and resuscitation were followed by equilibration periods. All subjects survived. A major/minor axis ratio of the Poincare plot that is greater than 5 is a useful index of shock.
Plasma potassium concentrations after severe controlled hemorrhage in inbred rat strains
Authors: Klemcke H. G. , Kheirabadi B. , Ryan K. L.
Published: June 2021

Arterial plasma potassium concentrations [K+]a increase after severe hemorrhage (Shires et al., Ann Surg 176:288, 1972)‐‐ which is consistent with decreased activity of ATP‐dependent Na/K ATPase (Darlington and Gann, J Trauma 58: 1, 2005)‐‐ and are significantly higher in non‐survivors vs survivors after hemorrhage (Torres et al., Am J Physiol 286: H1811, 2004). Eight‐fold differences in survival time were detected among 15 inbred rat strains after a controlled hemorrhage without resuscitation (Klemcke et al., Shock, in press, 2007). To assess potential mechanisms of these differences, [K+]a was measured at the beginning (basal) and end (final) of hemorrhage in those 15 inbred strains. Rats (n = 8–11/strain)were catheterized and, 24 hours later, 55% of the blood volume was removed during a 26 min period from conscious unrestrained animals. Rats were then observed for 6 hr or until death. Using average values for each strain, there were correlations between survival time and basal [K+]a (r= 0.52; P=0.04), final [K+]a (r= −0.70; P<0.01) and changes in [K+]a (final ‐ basal; r= −0.83; P<0.01). In support of these correlations, analysis of all strains via ANOVA revealed that longer‐lived strains had higher basal [K+]a, lower final [K+]a, and diminished changes in [K+]a (P<0.01) than did strains with shorter survival times. These results suggest genetic differences in mechanisms associated with both potassium homeostasis and hemorrhage‐induced alterations.
Alternative sources of cardiac norepinephrine (NE) transporter (NET)
Authors: Wehrwein Erica A , Bloch K , Spitsbergen J M , Yoshimoto M , Osborn J W , Kreulen D L
Published: June 2021

Cardiac norepinephrine (NE) is cleared from the neuroeffector junction by NE transporter (NET) and reuptake is impaired in many diseases. Cardiac NET is in sympathetic neurons and intrinsic cardiac adrenergic cells, and NE uptake is reduced by sympathetic denervation. However, we report substantial NET protein in the sympathetically denervated heart and thus examined alternative localization of NET in other nervous structures within the heart. We hypothesized that NET was in cardiac sensory and/or cholinergic neurons. Normal adult Sprague‐Dawley rats were used (n=7). NET mRNA and protein are found in sympathetic stellate ganglia and NET immunoreactivity (IR) is colocalized with tyrosine hyrdoxylase (TH)‐positive fibers in the heart. However, there are NET positive‐TH negative fibers. NET mRNA and protein are found in dorsal root ganglia; however, NET IR was not colocalized with calcitonin gene related peptide in sensory nerve fibers in the heart. NET mRNA is present and NET IR is colocalized with choline acetyl transferase‐positive intrinsic ganglia and fibers in the left atria. Thus, NET is found in sympathetic, sensory, and intrinsic cardiac ganglia, and in sympathetic and cholinergic fibers. In conclusion, there are alternative sources of cardiac NET protein aside from sympathetic nerve fibers that should be considered in the study of NET function. P01HL70687, AHA predoctoral fellowship (Wehrwein)
Hemorrhage induced inactivation of presynaptic group III mGluRs controls metaplasticity in circuits regulating fluid balance
Authors: Kuzmiski Brent J , Pittman Quentin J , Bains Jaideep S
Published: June 2021

In response to perturbations in body fluid homeostasis, noradrenaline (NA) is released in the paraventricular nucleus (PVN) of the hypothalamus to modulate neuroendocrine output. Here, we use whole‐cell patch clamp recordings from magnocellular neurosecretory cells in the PVN to show that bath application of NA (5 min, 100 μM) induces a robust, fast‐acting, long‐term depression (LTD) at glutamatergic synapses. This LTD is accompanied by a loss of presynaptic signaling at group III mGluRs. Depressed, but not naïve glutamatergic synapses exhibit a rapid presynaptic potentiation in response to high frequency stimulation. To determine if a physiological stressor that activates noradrenergic fibers to the PVN could induce similar changes in synaptic strength, slices were taken from rats that were hemorrhaged (under anesthesia, removal of 25% blood volume via cardiac puncture). In hemorrhaged rats, glutamatergic synapses displayed a reduced release probability and were unresponsive to the mGluR agonist, L‐AP4. High frequency stimulation of these synapses induced a rapid LTP. The ability of synapses to switch between depressed and potentiated states may be an important mechanism for filtering irrelevant activity (i.e. emotional inputs) from activity critical to controlling cardiovascular homeostasis. This work was supported by the Canadian Institutes of Health Research.
Macrophage enhances Neuronal Excitability: Implications for HIV‐1‐associated Neuronal Dysfunction.
Authors: Wang Wenwei , Xiong Huangui
Published: June 2021

Macrophages have been proposed to play an active role in HIV‐associated neuropathology and other neurodegenerative disorders. We hypothesize that macrophages alter neuronal excitability in HIV‐1‐infected brain and this alteration causes neuronal dysfunction. To test this hypothesis, we studied the influence of human monocyte‐derived macrophages (MDMs) and LPS stimulated MDMs(LPS‐MDMs)on rat cortical neuronal physiology in MDM and LPS‐MDMs‐neuronal co‐cultures using whole‐cell patch clamp techniques. When co‐cultured with neurons, LPS‐MDMs significantly increased spontaneous spikes in comparison with controls. Spike firing in response to constant current depolarizing injection, AP after hyperpolarization were significantly enhanced by LPS‐ MDMs. In addition, LPS‐MDMs decreased duration of APs and latency to spike firing. These results demonstrate that LPS‐MDMs, when co‐cultured with neuronal cells, alter neuronal passive membrane properties and enhance neuronal excitability. These alterations on neuronal physiology may result in neuronal dysfunction or injury as seen in HIV‐1‐infected brain.
BDNF protein exhibits a circadian rhythm in the hypoglossal, but not phrenic, motor nucleus
Authors: Guenther Courtney Helena , Dale‐Nagle Erica A. , Mitchell Gordon S.
Published: June 2021

BDNF is a key molecule in respiratory plasticity, particularly long‐term facilitation (LTF) following intermittent hypoxia (Baker‐Herman et. al., 2004). LTF is prominent in upper airway motor activity and may help to maintain upper airway patency during sleep, preventing the recurrence of obstructive apneas (Mahamed and Mitchell, 2007). Although BDNF exhibits a circadian rhythm within the suprachiasmatic nucleus (Liang et. al., 1998), it is unknown if BDNF exhibits circadian rhythms in regions of interest to respiratory control, such as respiratory motor nuclei. To test the hypothesis that BDNF protein exhibits circadian rhythms in the phrenic and hypoglossal (XII) motor nuclei, tissues were sampled from ventral cervical spinal segments (C3–C5) and XII nucleus in Sprague‐Dawley rats, (light/dark cycle: 6AM/6PM), at 6, 12, 18, and 24 hours. Results: BDNF protein exhibited a circadian cycle in the XII, but not phrenic, motor nucleus; BDNF protein levels were lower during the subjective night (12PM–5PM). At its lowest point (5PM–7PM), BDNF was 66 ± 11.5% (S.E.M.) lower than at 5AM‐7AM (p<0.05). The circadian BDNF rhythm may have important implications for the expression of respiratory plasticity in motor output, particularly to muscles controlling upper airway resistance. Thus, BDNF rhythms may contribute to the pattern of obstructive sleep apnea (Supported by NIH HL 80209).
Intrathecal administration of Vascular Endothelial Growth Factor (VEGF) elicits phrenic motor facilitation
Authors: Dale‐Nagle Erica Arden , Mitchell Gordon S
Published: June 2021

VEGF is a hypoxia sensitive growth factor historically viewed as a regulator of angiogenesis. However, recent evidence demonstrates that VEGF is expressed in phrenic motor neurons (Dale et al., FASEB J., 2006), and is neuroprotective for motor neurons (Storkebaum et al. Bioessays, 2004). Brain derived neurotrophic factor (BDNF) elicits phrenic motor facilitation by activating its high affinity receptor TrkB (Baker‐Herman et. al., 2004). Because VEGF receptors and TrkB are both receptor tyrosine kinases that act via common signaling pathways, we hypothesized that intrathecal VEGF administration would elicit phrenic motor facilitation similar to BDNF. We tested this in anesthetized, pump‐ventilated, vagotomized, and paralyzed, male Sprague‐Dawley rats prepared with intrathecal catheters at C4. Intrathecal VEGF (100ng with 0.1% BSA in artificial aCSF; n=3) increased integrated phrenic motor activity for at least 90 min post‐injection (30 min post‐injection: 63% ± 21% baseline; 60 min post‐injection: 84% ± 9%; 90 min post‐injection: 70% ± 14%; all p < 0.05). Phrenic motor facilitation was not observed in rats treated with intrathecal vehicle control (n=2). VEGF induced phrenic motor facilitation is similar in magnitude and time‐course to BDNF induced facilitation, providing the first evidence that VEGF may be an important factor of plasticity in respiratory motor neurons. Supported by NS057778 and HL80209.
Delta‐opioid receptor (DOR) activation prolongs respiratory motor output during oxygen‐glucose deprivation (OGD) in neonatal rat spinal cord in vitro
Authors: Freiberg Sara Marie , Gussick Megan Elizabeth , Johnson Stephen Mark
Published: June 2021

Severe reduction of blood flow to the brain results in oxygen‐glucose deprivation (OGD) and neuronal damage. DOR activation is neuroprotective during OGD in the cerebellum and cortex, but it is not known whether DOR activation protects spinal motor circuits that contribute to breathing. To address this question, a split‐bath in vitro neonatal rat brainstem/spinal cord preparation was used to record spontaneous respiratory motor output from cervical (C4–C5) and thoracic (T4‐T7) ventral spinal roots. A plastic barrier at spinal segment C1 allowed oxygenated solution to continuously bathe the brainstem while the solution on the spinal cord was switched from oxygenated to OGD (0 mM glucose, bubbled with 95% nitrogen/5% carbon dioxide) with or without DADLE (1.0 μM, DOR agonist). After OGD solution was applied to the spinal cord (n=7), cervical and thoracic motor output was abolished at 25.8 ± 2.0 and 26.4 ± 1.6 min, respectively. However, when DADLE was applied 10 min prior to and during spinal OGD solution application (n=8), cervical and thoracic motor output was abolished at 41.5 ± 4.9 min (p = 0.014) and 57.0 ± 4.2 min (p = <0.001), respectively. These data suggest that spinal DOR activation protects both cervical and thoracic respiratory motor output during OGD.
Irreversible damage to rostral pontine nuclei in adult awake goats does not alter the ventilatory regulation of eupneic PaCO2 and the ventilatory response to hypercapnia
Authors: Bonis Joshua M , Davis Suzanne , Kiner Tom , Krause Katie , Qian Baogang , Pan Lawrence , Forster Hubert
Published: June 2021

Controversies regarding the role of rostral pontine nuclei (RPN) in the control of breathing have prompted us to chronically implant microtubules into the rostral pons of 6 goats to assess the regulation of eupneic PaCO2 and the ventilatory response to hypercapnia following irreversible damage to RPN. All goats recovered uneventfully and 3 weeks after implantation PaCO2 and CO2 sensitivity were within normal limits. Whole body CO2 sensitivity was assessed by increasing inspired CO2 to 3, 5, and 7% at 5‐minute intervals, subsequent to breathing room air for 30 minutes. CO2 sensitivity was expressed as ΔVI/ΔPaCO2. Arterial blood was sampled during the control and min 4 of each hypercapnic level. As shown in the Table, CO2 sensitivity and resting PaCO2 were unaffected following sequential RPN damage (microtubule implantation, microdialysis probe insertion, and intra‐cranial ibotenic acid injection, respectively). We conclude that damage to RPN does not alter regulation of eupneic PaCO2 and the ventilatory response to hypercapnia.

CO2 sensitivity and resting PaCO2 are not significantly changed subsequent to RPN damage.

Pre‐implant
Post‐implant
Post‐microdialysis
Post‐ibotenic acid

CO2 sensitivity (l/min/mmHg)
2.24
2.25
2.08
2.30

Resting PaCO2 (mmHg)
40.24
39.57
38.67
40.00

No significant difference between variables by one‐way ANOVA (P<0.05). Supported by the Department of Veterans Affairs and NIH HL25739.
Respiratory response to lesioning of the hypothalamic paraventricular nucleus (PVN) with neuropeptide Y‐saporin (NPY‐SAP)
Authors: Wu Mingfei , Xu Guang , Mack Serdia O.
Published: June 2021

The PVN plays an integrative role in appetitive behavior, energy expenditure and autonomic function. NPY has a powerful orexigenic effect when injected into the PVN, which contains abundant NPY projections from the arcuate nucleus of the hypothalamus and brainstem. We selectively lesioned neurons in the PVN to determine the role of NPY in modulating respiratory drive. Young male rats were injected bilaterally with NPY‐SAP (50 ng/100 μl per side) neurotoxin or saporin (control vehicle). Body weight and temperature were similar (P>0.05) for both groups over an 8 week period after lesioning. Respiratory activity was measured in conscious, unrestrained animals. Under baseline conditions (breathing room air), NPY‐SAP rats maintained normal rhythmic breathing over the 8 week period. In the treated group, the percent change in the tachypneic response to acute hypoxia (8% O2) was elevated significantly (P<0.05) above the response for the control group starting at 2 weeks after lesioning. NPY‐SAP animals exposed to 5% CO2 initially had a greater increase in respiratory frequency for 4 weeks after treatment, followed by a decline below the levels for the controls 8 weeks after lesioning. These results suggest that a subset of NPY‐responsive cells in the PVN is involved in modulating ventilatory chemoresponses. Supported by NIH NS45859 and NINDS/NCRR U54NS39407
Mechanical stretch induces MCP‐1 production via aTNF‐alpha/cPLA2‐dependent pathway in human proximal tubular (HK‐2) cells
Authors: Alexander Larry D
Published: June 2021

Increased tumor necrosis factor (TNF)‐alpha and monocyte chemoattractant protein‐1 (MCP‐1) production in obstructive nephropathy are considered major events leading to persistent inflammation in the kidney, but the molecular mechanisms involved in this process are poorly understood. This study was designed to investigate the effect of mechanical stretch on the production of cytokines in kidney epithelial cells. The levels of TNF‐alpha and MCP‐1 were significantly increased by cyclic stretch and the exogenous application of arachidonic acid. Prostaglandin (PG) E2 production and cytosolic phospholipase A2 (cPLA2) activity was also increased by cyclic stretch. Stretch‐ induced TNF‐alpha and MCP‐1 production was blocked by inhibition of cPLA2, cytochrome p450 epoxygenase, and cyclooxygenase, but not lipoxygenase, suggesting that stretch‐ induced TNF‐alpha and MCP‐1 production is dependent on synthesis of arachidonic acid and its subsequent metabolism to epoxyeicosatrienoic acids and/ or prostaglandins. Furthermore, TNF‐alpha blockade resulted in significant inhibition of both, stretch‐ and arachidonic acid‐ induced MCP‐1 and PGE2 production. Overall, these findings suggest that TNF‐alpha mediates stretch‐ induced proximal tubular MCP‐1 production through cPLA2/arachidonic acid activation and that stretch induced pro‐inflammatory effects could be regulated, in part, by inhibition of prostaglandins and/ or epoxyeicosatrienoic acids production and function.
The small G protein Ras and Ras/MAPK/ERK pathway are up‐regulated in the rostral ventrolateral medulla of stroke‐prone spontaneously hypertensive rats
Authors: Kishi Takuya , Hirooka Yoshitaka , Sunagawa Kenji
Published: June 2021

Previous reports suggested that Angiotensin II in the rostral ventrolateral medulla (RVLM) caused the presser response through the activation of activated mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) 1/2, which in turn determines the sympathetic nerve activity (SNA). However, it remains unknown whether small G protein Ras, upstream of MAPK and ERK, in the RVLM is up‐regulated in hypertensive rats. The aim of this study was to determine whether Ras and Ras/MAPK/ERK pathway in the RVLM are up‐regulated in stroke‐prone spontaneously hypertensive rats (SHRSP) in pre‐hypertensive and hypertensive states. In SHRSP and age‐matched Wistar‐Kyoto (WKY) rats, we sampled the RVLM tissues by punched‐out technique. In hypertensive states, SHRSP had significantly higher expression of Ras (+28%, n=8, p<0.01), phosphorylated p38 MAPK (+33%, n=8, p<0.01) and ERK1/2 (+36%, n=8, p<0.01) than WKY, and significantly higher activity of Ras (+38%, n=8, p<0.01). In pre‐hypertensive states, whereas the expressions of Ras, phosphorylated p38 MAPK and ERK1/2 were not different between SHRSP and WKY, the activity of Ras was significantly higher in SHRSP than in WKY. (+42%, n=8, p<0.01) In conclusion, these results suggest that Ras and Ras/MAPK/ERK pathway in the RVLM are up‐regulated in the SHRSP, and that this pathway might be involved in the pathogenesis of hypertension.
Receptor mediated endocytosis; a possible pathway for specific immunity in the Eastern Oyster, Crassostrea virginica
Authors: Gohad Neeraj Vijay , Mount Andrew S
Published: June 2021

The evolution of challenge specific long term immunity in the Mollusca and other invertebrates is intriguing in that it implies a level of cellular differentiation and immune complexity previously reported only in vertebrates. Correlative confocal laser scanning and scanning electron microscopy were employed to study receptor mediated endocytosis. We report a class of dendritic‐like cells found in the hemolymph of the oyster which appear to function like antigen presenting cells. These cells proteolytically cleave fluorescently labeled but self‐quenched ovalbumin into fluorescencent peptides, via Fc receptor mediated endocytosis. Oyster dendritic‐like cells comprise 4% of the circulating hemocytes and are distinguished from other cells by the presence of β‐adrenergic receptors on their plasma membranes. Research support: Authors would like to thank Office of Naval Research for the research support.
The Influence of Sex and Obesity Phenotype on Meal Fatty Acid Metabolism Before and After Weight Loss
Authors: Santosa Sylvia , Hensrud Donald D , Votruba Susanne B , Jensen Michael D
Published: June 2021

Objectives: To determine whether meal fatty acid (FA) metabolism differs by sex and obesity phenotypes and to examine how weight loss (WtL) through diet and exercise affects meal FA trafficking.

Methods: Before WtL (study 1) 13 obese men (ObM), 9 upper body obese (UbO) and 8 lower body obese (LbO) women consumed a standard meal containing [3H]triolein followed by subcutaneous abdominal and gluteal fat biopsies. Indirect calorimetry was measured by 5 h substrate oxidation. Whole/regional fat distribution was measured using DXA and CT scan. Participants then enrolled in a structured WtL program, after which 23 returned for study 2.

Results: There were no differences (p=0.32) in meal FA oxidation between groups. Abdominal uptake of meal FA in LOb was greater than in UOb (p=0.026) and ObM (p<0.001). LOb took up more meal FA in the lower body than ObM (p=0.001). Participants who returned for study 2 lost 14 ± 1.1 kg. After WtL, uptake of meal FA in upper body subcutaneous fat increased (p=0.028) in UOb and ObM (p=0.046) and decreased in lower body fat (p=0.025) in ObM. WtL led to reduced 5 h postprandial total fat oxidation and increased 24 h meal FA oxidation.

Conclusion: Changes in uptake of meal FA after WtL followed a pattern indicative of body fat distribution maintenance. These results highlight the potential importance of meal fat uptake in modulating body fat distribution in obesity.

Funding: NIH RO1 DK45343, DK50456 and R00585.
Amyloid‐beta stress test in patients with Alzheimer disease
Authors: Fiala Milan , Zaghi Justin , Liu Philip T. , Rosenthal Mark
Published: June 2021

The innate immune system of patients with Alzheimer disease (AD) is defective in clearance of amyloid‐beta (Ab) and transcription of genes important for phagocytosis including MGAT‐3 and Toll‐like receptors. Control subjects’ macrophages clear Ab through phagocytosis and transport into lysosomes, whereas AD patients’ macrophages bind but do not endocytize Ab. Immune clearance of Ab by monocytes is a complex process involving expression of surface proteins and dendrites on monocytes and aggregation of monocytes around Ab. We tested peripheral blood monocytes of patients with sporadic AD by an “Ab stress test” involving exposure to Ab in overnight culture and testing of Ab phagocytosis (controls > patients by fluorescence microscopy, P=0.001; by flow cytometry, P=0.011), expression of two surface adhesion proteins (controls > patients, P =0.001), and propensity to apoptosis from fibrillar Ab (patients >controls, P=0.028). We are testing age‐matched AD and control subjects to determine the stage of the disease when immune defects are recognized. “Ab stress test” shows immune biomarkers and might assign subjects into AD risk categories. Supported by Alzheimer's Association.
Apolipoprotein E4 Genotype Influences RAGE and LRP‐1 Expression
Authors: Donahue John Edward , Meitner Patricia A. , Tavares Rosemarie , Vitek Michael P. , Jackson Cynthia L.
Published: June 2021

The presence of the ε4 allele of the apolipoprotein E gene (APOE4) increases the risk of developing Alzheimer's disease (AD) through unclear mechanisms. In severe AD, there is upregulation of the receptor for advanced glycation endproducts (RAGE), which transports amyloid‐β (Aβ) into the brain, and concurrent downregulation of the low‐density lipoprotein receptor‐related protein (LRP)‐1, which transports Aβ out of the brain, at the blood‐brain barrier. This causes an elevated brain Aβ burden in AD. (Total LRP‐1 expression increases in severe AD brains because LRP‐1 colocalizes with apoE protein and Aβ in senile plaques.) This study demonstrates about a 7‐fold increase in RAGE and 3‐fold increase in LRP‐1 mRNA expression in 52‐week‐old transgenic knockin mice that are homozygous for the human APOE4 gene, compared to 52‐week‐old mice that are homozygous for APOE3. (In 26‐week‐old APOE3/3 and APOE4/4 mice, RAGE and LRP‐1 expression are similar to the 52‐week‐old APOE3/3 mice.) This suggests that APOE genotype can influence both RAGE and LRP‐1 expression with aging and is a potential mechanism whereby the presence of APOE4 is a risk factor for developing AD pathology. (Support: NIH grant AG029147)
Experimental gliomas in mice using the Sleeping Beauty (SB) transposon system: neuropathologic aspects
Authors: Rodriguez Fausto J , Collier Lara S , Green Laura E , Bender Aaron , Giannini Caterina , Largaespada David A , Jenkins Robert B
Published: June 2021

The SB transposon system has been used as an insertional mutagen to study tumor development. This novel system involves the mobilization of a transposon (e.g. T2/onc) by a tranposase source (e.g. Rosa26‐SB11) in transgenic mice. We serially sectioned FFPE brains from 154 mice and identified 15 infiltrating gliomas that morphologically resembled small cell astrocytomas, as well as one PNET. The gliomas involved cerebral hemispheres (n=8) or posterior fossa (n=7). The tumors demonstrated remarkable infiltration of underlying brain parenchyma with secondary structuring (i.e. subpial, perivascular and perineuronal aggregation); round to oval cells with irregular nuclear contours, scant cell processes and variable mitotic activity (median 4/10 hpf, range 2–9); and pseudopalisading necrosis (n=5). Endothelial proliferation and calcification were absent. Immunohistochemical stains for GFAP and S100 strongly labeled reactive astrocytes, but minimal immunoreactivity was also seen in occasional small tumor cells. In 14 of 15 mice the genotype was T2/onc/SB11 positive; three mice were p19Arf+/−, one mouse was p19Arf−/− and one was Blm−/−. The PNET filling the third ventricle was identified in a control mouse homozygous mutant for p19Arf, but transgenic only for the T2/Onc concatomer. This tumor, in contrast to the gliomas, demonstrated uniform synaptophysin staining and did not label with GFAP or S100.These results demonstrate that the SB transposon system is a valid model to study gliomagenesis in mice.
Effects of dietary bioactive polyphenols on iron transport in human intestinal Caco‐2 cells
Authors: Kim Eun‐Young , Ham Soo‐Kyung , Shigenaga Mark , Han Okhee
Published: June 2021

There is persuasive epidemiological evidence that consumption of bioactive dietary polyphenolic compounds, which are present in fruits, vegetables, some types of grain, red wine and tea, promote human health. Bioactive dietary polyphenolic compounds reportedly have a wide range of effects in vivo and vitro, including metal chelating activities. Tea and other beverages rich in phenolic compounds were shown to inhibit the absorption of non‐heme iron. Because iron deficiency is the most common nutritional deficiency and the mode action of these compounds includes chelation of metals such as iron, it is prudent to examine the effects of dietary bioactive polyphenols on iron absorption. Initially, we examined effects of epigallocatechin‐o‐gallate (EGCG), a major polyphenolic component of a green tea, and grape seed extract (GSE) on intestinal iron transport. The fully differentiated Caco‐2 cells grown on microporous membrane inserts were used to examine effects of bioactive dietary polyphenolic compounds on the absorption of non‐heme iron. The transepithelial transport of 55Fe from the apical compartment into the basolateral compartment was 17.6 ± 2.2 pmol/mg protein during 3h incubation. Addition of 10‐fold molar excess of EGCG and GSE decreased 55Fe transport by 98 ± 0.3 and 97.1±0.2 %, respectively. These results demonstrate the inhibitory influence of dietary bioactive polyphenols on the absorption of non‐heme iron.
TES7, a monoclonal antibody targeting B7‐H3, potently inhibits Hs‐700T growth in vivo.
Authors: Liang Tony Wei‐yen , Roberts Penny E , Young Peter , Li Pam , Santos Mary Ann , Chen Francine , Mather Jennnie P , Vehar Gordon A
Published: June 2021

Human B7‐H3, a member of the B7 family of costimulator molecules, is a ∼110kDa type I membrane protein with 4 extracellular Ig like loops (IgV1‐IgC1‐IgV2‐IgC2), a short cytoplasmic tail, and a putative isoform with only 2 extracellular Ig like loops (IgV2‐IgC2). In this report, we present and characterize TES7, a monoclonal antibody specific to human B7‐H3. We map TES7 binding to the IgV1‐IgC1 portion of B7‐H3, and demonstrate TES7/B7‐H3 interaction to be density and receptor clustering sensitive. Immunohistochemical studies of TES7 show differential staining on normal versus cancerous human tissues (normal pancreas, lung, liver, kidney, and heart were negative, membrane staining observed on colon epithelia; positive staining on 4/4 lung cancer, 4/4 colon cancer, and 6/13 pancreatic cancer samples; 4/4 breast cancer and 3/3 prostate cancers samples were positive). Using TES7 in vivo, we demonstrate growth inhibition of subcutaneously implanted Hs‐700T cells. The data presented are the first demonstration of disease‐associated reorganization of B7‐H3 surface expression, and presents TES7 as a novel tool for the study of B7‐H3 biology and as a potential cancer therapeutic.
A neurovascular niche for neurogenesis after stroke
Authors: Carmichael S. Thomas
Published: June 2021

Stroke causes localized tissue destruction within the infarct. Stroke also induces a limited degree of regeneration in the tissue that borders the infarct, including a process of post‐stroke neurogenesis. In post‐stroke neurogenesis, newly born, immature neurons (neuroblasts) migrate long distances from the subventricular zone to areas of damage. Neuroblasts migrate into these areas of damage in close association with remodeling, angiogenic blood vessels. Inhibiting angiogenesis after stroke blocks neurogenesis. During the time period of neuroblast migration angiogenic blood vessels secrete SDF‐1 and angiogpoietin‐1. Gain and loss of function studies show that these molecules are tropic for migrating neuroblasts in vivo after stroke. This data identifies a neurovascular niche after stroke in which neurogenesis is causally linked to angiogenesis through specific vascular growth factors and chemokines. This data follows from studies of the neural stem cell niche and neuroblast migration in the normal animal to highlight vascular‐neuronal interactions in both normal and regenerative brain function. Supported by NS053957, the Larry L Hillblom Foundation and AHA 0555013Y.
Antigen‐induced Lymph Node Remodeling: LVs, HEVs and Conduits
Authors: Stranford Sharon A , Liao Shan , Bentley Kevin , Ruddle Frank H. , Ruddle Nancy H
Published: June 2021

Secondary lymphoid organs are the sites of antigen encounter with naïve, antigen‐specific lymphocytes, and as such are crucial to the development of acquired immunity. In lymph nodes (LNs), this process is facilitated by the convergence of high endothelial venules (HEVs) and lymphatic vessels (LVs) delivering lymphocytes and tissue‐derived antigen, respectively. Conduits, a third system of channels, may directly connect the afferent lymphatics with the abluminal surface of HEVs, facilitating rapid communication between the two. We investigated the relationship of these structures in draining LNs during antigen encounter. Mice transgenic for HEC‐6ST (an HEV unique gene) driving eGFP expression were skin painted with oxazalone. Four days later, draining LNs were removed and sections were stained for LV‐, HEV‐ and conduit‐specific markers. At day 4, expression of HEC‐6ST was suppressed, with concomitant enhancement of MAd‐CAM‐1 expression, a marker of an immature HEV phenotype. Surprisingly, the endothelial cells of some vessels expressed both Lyve‐1 and PNAd, or displayed a mixture of HEC‐6ST+ and Lyve‐1+ cells. The ER‐TR7+ conduit network permeated the T cell areas and surrounded HEVs. These data demonstrate that Hec6st‐egfp transgenic mice can be used to evaluate vascular interactions in draining LNs. Supported by NIH CA16885 and DK50731 (NHR) and NIH R15 AI051674‐02 (SAS).
Build‐your‐own paper model of the pelvic diaphragm and perineum
Authors: Downie Sherry A.
Published: June 2021

One of the consistent challenges in teaching the anatomy of the pelvis and perineum is conveying the three‐dimensional relationships of the bony pelvis, pelvic diaphragm and the structures and spaces of the urogenital region. Based on the many questions that students ask during dissection of the pelvis and perineum, two‐dimensional media such as textbooks and PowerPoint presentations, regardless of the quality or quantity of images, are not universally effective in helping students generate an anatomically accurate and clinically relevant understanding of the region. Commercial models of the pelvis and perineum are available that provide the third dimension lacking in texts and PowerPoints but they are expensive and accessible to students only on a limited basis. In addition, commercial models do not reproduce the distinct layers and spaces of the perineal pouches that are so difficult and essential for students to understand. Dr. D.'s Super‐Duper Simple Paper Pelvis Kit is an inexpensive, easy to assemble and portable three‐dimensional model that can be given to each student to construct prior to dissection of the pelvis and perineum. Through the act of assembling the kit students create for themselves a model that replicates the three‐dimensional relationships of the bony pelvis, pelvic diaphragm and perineal pouches with their fascias, spaces and contents, thus adding clarity and understanding to the often problematic perineal dissections. Inspired students may be motivated to build their own pelvic organs to add to the model!! A large scale model, ideal for group demonstrations, is also available.
Learning human anatomy through comparative vertebrate anatomy
Authors: Smith Gregory R.
Published: June 2021

Studying comparative vertebrate anatomy gives us insights to the design of these animals. Vertebrates exhibit anatomical adaptations to their various and unique environments. An appreciation of human design and function is gained when we compare ourselves to examples of other vertebrates that are considered specialists in the animal kingdom.
A tale of two reverse transcriptases – assembly, structural dynamics and function of telomerase and HIV reverse transcriptase
Authors: Zhuang Xiaowei
Published: June 2021

Telomerase is a cellular ribonucleoprotein (RNP) that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes. It is a specialized reverse transcriptase (RT) that uses sequence encoded in its own RNA component as a template to synthesize telemetric DNA repeats. The RT of human immunodeficiency virus (HIV) is a viral protein that catalyzes an orchestrated series of reactions, including RNA‐directed DNA synthesis, DNA‐directed DNA synthesis and DNA‐directed RNA hydrolysis, to convert the single‐stranded RNA genome of HIV into double‐stranded DNA for host‐cell integration. Using single molecule FRET, we have investigated the assembly pathway, structural dynamics and structure‐function relation of these two RT systems. For both RT‐nucleic acid substrate complexes, novel assembly and structural dynamics were discovered that are critical for the enzymatic functions of these systems.
Serine/Threonine Acetylation: Competing for Hydroxols
Authors: Orth Kim
Published: June 2021

Recent studies on a bacterial virulence factor, YopJ of Yersinia, led to the discovery that serine and threonine residues can be modified by acetylation. As YopJ uses a unique mechanism of inhibition (acetylation that directly competes with phosphorylation of serine and threonine residues), we are able to use this inhibitor to analyze mechanisms involved in the activation of the kinases that are sensitive to YopJ's inhibition. Studying other YopJ‐like acetyltransferases reveal different ways bacterial effectors can manipulate eukaryotic signaling systems by acetylating signaling molecules. Although the identification of the machinery used for the addition and removal of acetyl groups on Ser or Thr residues is in its infancy, the enzymes thus far studied provide early insight into the mechanism of this newly discovered posttranslational modification, and hint at its potential importance. Molecular mechanisms and future challenges in analyzing serine and threonine acetylation and deacetylation are also discussed.
Manipulating and Detecting a Protein's Energy Landscape
Authors: Marqusee Susan
Published: June 2021

We have developed a novel method to explore the single molecule behavior of proteins in the low force regime of the laser tweezers. Individual protein molecules are tethered between two polystyrene beads using two dsDNA molecules, which are in turn attached to the protein through unique engineered cysteine residues. One bead is held by suction on the tip of a pipette while the other is held in the optical trap. The force and distance between the beads is varied by moving the bead on the pipette relative to the bead in the trap. The DNA handles can be attached to any exposed cysteine residues, allowing a variety of pulling geometries to be used to investigate the anisotropy of the protein energy landscape. The low spring of laser tweezers enabled us to directly monitor low force refolding events and fluctuations between different molecular structures at quasi‐equilibrium conditions. Data exploring the behavior of different protein protein topologies, different pulling geometries and the relationship to bulk solution studies of the energy landscape will be presented.
Integration of mechanical and chemical signals in cell protrusion
Authors: Danuser Gaudenz
Published: June 2021

Cells move by forces associated with the assembly of an actin cytoskeleton network which are balanced by adhesive coupling of the cytoskeleton to the extracellular domain and counteracted by cytoskeleton contraction powered by molecular motors. The forces are orchestrated in space and time by feedback signals that process the mechanical and chemical states of the cell. Our goal is to decipher the largely unknown design principles and modes of operation of this complex molecular system. We developed a number of techniques to map out from high‐resolution live cell light microscopy the structural dynamics of the actin network, the contraction, adhesion, and propulsion forces transmitted through the network, and the activation of signaling molecules implicated in the regulation of actin cytoskeleton dynamics. Computational methods are being designed to integrate this data in a global model of the coupled regulatory and mechanical pathways mediating morphogenic events like cell protrusion. A corner stone of the framework is the concept of exploiting constitutive random fluctuations of pathway component activities observed over many experiments to infer from the similarity of fluctuations within and between cells the hierarchical relationships between pathway components.
Membrane biogenesis induced by the unfolded protein response
Authors: Brewer Joseph W. , Bommiasamy Hemamalini , Sriburi Rungtawan , Jackowski Suzanne
Published: June 2021

A hallmark of specialized secretory cells is an elaborate network of endoplasmic reticulum (ER), a factory for synthesis and assembly of secretory pathway proteins. To delineate mechanisms underlying ER biogenesis, we have investigated the unfolded protein response (UPR), a multifaceted signaling system that allows cells to cope with increased demand on the protein folding capacity of the ER. A transcriptional activator, XBP‐1(S), generated by UPR‐mediated splicing of Xbp‐1 mRNA is necessary and sufficient for proliferation of rough ER. XBP‐1(S)‐induced ER biogenesis in fibroblasts involves elevated production of phosphatidylcholine (PtdCho), at least in part due to increased synthesis of the rate‐limiting enzyme (choline cytidylyltransferase) in the CDP‐choline pathway of PtdCho synthesis. In parallel, XBP‐1(S) drives expression of many genes encoding proteins that function in the ER, thereby coordinately increasing supplies of ER proteins and membrane lipids. Like XBP‐1(S), overexpression of the UPR transcriptional activator ATF6 enhances lipid biosynthesis and ER abundance, yet these two UPR factors differ in their effect on expression of lipid metabolic genes. These data reveal links between the UPR, lipid biosynthesis and mechanisms that orchestrate ER biogenesis. [supported by NIH Grants GM 61970 (J.W.B.), GM 45737 (S.J.), T32 AI007508 (H. B.) and by ALSAC]
Signal transduction by stress‐activated MAP kinases
Authors: Davis Roger J
Published: June 2021

The JNK group of stress‐activated MAP kinases consists of ten protein kinases that phosphorylate the NH2‐terminal activation domain of c‐Jun on Ser‐63 and Ser‐73 causing increased transcriptional activity. JNK protein kinase activity is increased in response to treatment of cells with pro‐inflammatory cytokines or exposure to environmental stress. Activated JNK is phosphorylated on Thr and Tyr within the tripeptide motif Thr‐Pro‐Tyr located in kinase sub‐domain VIII. Mutational analysis demonstrates that JNK activation requires the phosphorylation of both Thr and Tyr within this motif. This phosphorylation is mediated by dual specificity protein kinases, including MKK4 and MKK7.

The function of the JNK signaling pathway has been studied using a combination of biochemical and genetic approaches. Genetic analysis of JNK signaling in Drosophila demonstrates that JNK is required for early embryonic morphogenesis. Similarly, disruption of the JNK signaling pathway in mice using homologous recombination demonstrates that JNK is required for embryonic viability. In contrast, mice with genetically engineered selective defects in JNK signaling are viable, but exhibit changes in stress‐induced gene expression and apoptosis. These studies provide insight into the role of the JNK stress‐activated MAP kinase pathway in the cellular response to environmental stress, including apoptosis and cell survival.
Discovering adult stem cell expansion technologies
Authors: Sherley James L.
Published: June 2021

An over‐arching principle of our research is active integration of basic and applied investigations by design. Most recently, we have applied this principle to the challenge of enabling future adult stem cell‐based medical therapies. A major barrier to development of effective cellular therapies is difficulty producing functional tissue‐specific adult stem cells routinely and in large quantity. We addressed this problem by investigating molecular mechanisms in engineered cultured cell models that display the unique cell kinetics function of adult stem cells, asymmetric self‐renewal. Asymmetric self‐renewal, which functions to limit adult stem cell number in tissues, poses the major barrier to their expansion in culture. In the model cell studies, we discovered a p53‐dependent biochemical pathway that governs whether the self‐renewal kinetics of cells is asymmetric or symmetric. Whereas asymmetric self‐renewal maintains a constant adult stem cell number, symmetric self‐renewal leads to their rapid exponential expansion. Based on this discovery, we developed a new concept called “suppression of asymmetric cell kinetics” (SACK) for expansion of adult stem cells in culture. Recently, this concept was reduced to practice for expansion of cells with properties of adult stem cells from normal human liver. Based on the universality of asymmetric self‐renewal by adult stem cells, we anticipate that the SACK method will be effective for routine production of adult stem cells from diverse human tissues.
Energy Density and Weight Change in a Long‐Term Weight‐Loss Trial
Authors: Flood Andrew , Mitchell Nathan , Jaeb Melanie , Finch Emily A. , Laqua Patricia S. , Walsh Ericka M. , Hotop Annie , Langer Shelby , Levy Rona L. , Jeffery Robert W.
Published: June 2021

Identifying dietary factors that contribute to weight loss is an important task given the health risks linked to obesity and the difficulty in achieving weight loss among obese individuals. This study examined the effect of lowered energy density on weight loss. Subjects were 213 men and women with BMI of 30–39 kg/m2 and without chronic illness who were drawn from a randomized trial evaluating behavioral modifications for achieving long‐term weight loss. All subjects completed a 62‐item food frequency questionnaire at baseline and at 6, 12, and 18 months of follow‐up. Pearson correlation coefficients for BMI and energy density of diet (kcals/g of solid food) indicated no association at baseline (r=0.0039, p=0.95). At 6 months of follow‐up BMI had decreased an average of 2.23 kg/m2, and we observed a strong positive correlation between change in energy density and change in BMI (r=0.35, p<0.0001). Changes in energy density between 6 and 12 months of follow‐up were correlated with changes in BMI, but with smaller weight loss, we also observed a lower correlation (r=0.16, p=0.04). Between 12 and 18 months average change in BMI was essentially 0, and during this time there was no correlation between change in energy density and change in BMI (r=0.017, p=0.89). From these data we conclude that during periods of active weight loss BMI decreased the most among those who also had the greatest decrease in dietary energy density.