bluuebunny/crossref_metadata_2025 huggingface dataset:
PP1 and PP2a phosphatases can modulate AQP2 endocytosis
Authors: Moeller Hanne B. , Aroankins Takwa S. , Fenton Robert A.
Published: June 2021

Membrane accumulation of aquaporin‐2 (AQP2) is regulated by phosphorylation at S256 and S269. Our previous studies showed that mimicking phosphorylation at S256 and S269 (256D‐ and 269D‐AQP2) decreased internalization rates of AQP2 in MDCK cells. In cell lines expressing double mutated AQP2, 256D/269A‐AQP2 and 256D/269D‐AQP2 were predominantly located in the plasma membrane, whereas 256A/269A‐AQP2 and 256A/269D‐AQP2 had an intracellular localization. This suggests that pS256 is dominant for determining AQP2 localization. Cells expressing 256D/269A‐ or 256D/269D‐AQP2 had decreased AQP2 internalization compared to wt‐AQP2; with 256D/269D‐AQP2 being internalized slowest. To examine pS269 and pS256 in a dynamic system, wt‐AQP2 cells were treated with the phosphatase inhibitors Okadaic Acid and Calyculin A. Treatment decreased the rate of AQP2 endocytosis in response to forskolin removal. In parallel, pS269‐AQP2 levels were greatly increased and prolonged compared to control, whereas only minor changes in pS256‐AQP2 was observed. To examine if phosphorylation at S269 regulates AQP2‐ ubiquitination at K270, immunoprecipitations of N‐terminal flag‐tagged wt‐AQP2, 269A‐AQP2, and 269D‐AQP2 were assessed for ubiquitination. All AQP2 forms were ubiquitinated following either forskolin or combined forskolin and TPA treatment.
Hydrogen peroxide selectively increases paracellular leak pathway permeability of renal epithelial cells
Authors: Janosevic Danielle , Rohring Victoria , Amsler Kurt
Published: June 2021

Increased paracellular solute flux is observed in several renal pathogenic processes characterized by oxidative stress. We undertook a study to examine the effect of H2O2 treatment, as a model of oxidative stress, on paracellular permeability of two renal epithelial cell lines, LLC‐PK1 and MDCK. Both the pore pathway (small ions; TransEpithelial Resistance (TER)) and leak pathway (large solutes; calcein flux) were monitored. MDCK monolayer calcein flux was much lower than LLC‐PK1 monolayer calcein flux. In contrast, MDCK monolayer TER was about 2‐fold lower than LLC‐PK1 monolayer TER. Sublethal H2O2 produced a concentration‐dependent increase in calcein flux in both renal cell lines. In contrast, TER only decreased at H2O2 concentrations that increased cell death. H2O2 treatment did not produce a marked change in total contents of multiple tight junction proteins nor in their distributions between Triton X‐100‐soluble and –insoluble fractions, although treatment may have produced a modest decrease in Triton X‐100‐insoluble ZO‐2 content. Surprisingly, occludin overexpression impaired and occludin knockdown modestly enhanced the ability of H2O2 to increase transepithelial calcein flux. H2O2 treatment produced a transient activation of src Family Kinases (SFKs). SFK inhibitors, but not inactive analogs, attenuated the H2O2‐induced increase in calcein flux. (Supported by institutional funds)
ROS scavenging mimics anoxia by enhancing GABA receptor‐mediated electrical suppression in anoxia‐tolerant turtle cortex
Authors: Hogg David William , Pamenter Mathew Edward , Buck Leslie Thomas
Published: June 2021

In anoxic mammalian brain, ATP dependent ion pumping is compromised and the consequent loss of membrane ion gradients elicits hyper‐excitability and cell death. This does not occur in the western painted turtle brain, where γ‐aminobutyric acid (GABA) concentration increases 80 fold and electrical activity decreases 75–95%. During anoxia, electrical suppression results from increased GABA receptor (GABAR)‐mediated postsynaptic currents (PSC) that shunt excitatory current, thereby avoiding excess AP firing. The signaling pathway responsible for initiating GABAergic neuroprotection is unknown but could involve decreases in the concentration of reactive oxygen species (ROS) that occurs following onset of anoxia. Using whole‐cell and perforated patch clamp techniques we determined that, similar to anoxia, ROS scavenging caused: whole cell conductance to increase from 4.5 ±0.4 to 6.6 ±1.1 nS; AP threshold to depolarize from 45 ± 4.2 to 24 ± 2.2 mV; and GABAR‐mediated currents to double in amplitude from 237.8 ± 81.6 to 449.8 ± 136.9 pA. Pharmacological anoxia (normoxic plus cyanide) caused a similar increase in GABAR current amplitude to 407.8 ± 38.5 pA and this was prevented by ROS donors (hydrogen peroxide or xanthine oxidase/xanthine). We conclude that a decrease in mitochondrial ROS production is the signal that increases GABAR currents during anoxic stress. Research supported by OGS scholarship to DWH, NSERC postdoctoral fellowship to MEP and an NSERC Discovery and Accelerator Grant to LTB.
Using synchrotron X‐ray imaging to visualize tracheal changes during development in American locusts
Authors: Donohue Mary C. , Greenlee Kendra J. , Lee Wah-Keat , Kirkton Scott D.
Published: June 2021

During their eight‐week development, Schistocerca americana grasshoppers hatch as 15 mg 1st instars and grow into nearly 3 g adults. It has been suggested that larger insects may have more problems with O2 delivery. In support of this idea, S. americana CO2 emission rates only scale with body mass0.76 during development, and larger, older grasshoppers fatigue more rapidly during repeated jumping. Furthermore, the jumping muscle of larger, older grasshoppers relies more on anaerobic metabolism. To test the hypothesis, that larger, older grasshoppers have decreased O2 delivery, we used synchrotron X‐ray imaging to visualize tracheal air sac compressions in the abdomen, proximal femur, and distal femur of developing S. americana in 21% and 5% O2. For all ages, hypoxia significantly increased air sac ventilation rates throughout the body. There was no effect of body size across development on ventilation rates of abdominal or distal femoral air sacs in either 21% or 5% O2. However, larger, older grasshoppers had ventilation rates in the proximal femoral air sacs that were 70% lower in both 21% and 5% O2 than those of smaller, younger animals. The reduced ventilation rate of femoral air sacs in larger grasshoppers provides a respiratory mechanism that explains their greater reliance on anaerobic metabolism and also supports the hypothesis that they have more problems with O2 delivery. (Support provided by NSF, NIH, US DOE).
Miner1, mutated in Wolfram Syndrome, is an endoplasmic reticulum protein that regulates cellular redox status and Ca2+ homeostasis
Authors: Wiley Sandra E , Andreyev Alex , Perkins Guy , Dixon Jack E , Murphy Anne N
Published: June 2021

Miner1 (Cisd2) is a redox‐active 2Fe2S cluster protein linked to the development of Wolfram Syndrome, an untreatable disease associated with diabetes, blindness, and deafness. Using mouse embryonic fibroblasts derived from Miner1 deficient mice, we demonstrate that loss of Miner1 leads to endoplasmic reticular (ER) stress and the induction of the unfolded protein response. Miner1 has a profound effect on Ca2+ homeostasis, leading to a depletion of ER Ca2+ stores and an increase in mitochondrial Ca2+ load. These findings were associated with an increased rate of ATP utilization in intact cells, yet a slower growth rate, suggesting futile energy cycling. Furthermore, we observed ultrastructural alterations in the mitochondria lacking Miner1, and an increase in maximal O2 consumption without a change in the level of mitochondrial uncoupling. The cellular pyridine nucleotide and sulfhydryl redox status were shifted to a more oxidized state. Treatment with the antioxidant N‐acetylcysteine reversed most of the abnormalities in the Miner1 deficient cells. Thus, we establish Miner1 as a protein that regulates the redox status of the cell, and propose that Wolfram Syndrome is fundamentally a disease resulting from oxidative stress. Supported by the American Diabetes Assoc 1‐08‐RA‐139, and NIH 4R42DK081298.
LPLRFamide causes anorectic effects associated with the brain stem in rats
Authors: Webster Rebekah Ireland , Cline Mark
Published: June 2021

We investigated the effects of LPLRFamide on appetite‐associated processes using Sprague Dawley rats. LPLRFamide is a member of the RFamide family, a group of neurotransmitters that both stimulate and reduce food intake across many species. We previously showed that intracerebroventricular injection of LPLRF caused decreased feeding in chicks and we hypothesized that this would also be the case in rats. Rats were fasted for 24 hours and those injected with 7 and 15 nmol LPLRFamide, through indwelling guide cannulas aimed at the left lateral ventrical, significantly decreased feeding up to 40 minutes post injection. To investigate the central mechanisms of this effect, 24‐hour fasted rats were injected with 15 nmol LPLRFamide and 30 minutes following treatment, the number of c‐Fos immunoreactive cells was quantified in hypothalamic areas including the dorsomedial, ventromedial, and paraventricular nuclei, as well as the lateral hypothalamic area, and the dorsal motor nucleus (10N) and nucleus of the solitary tract (NTS) of the brainstem. There were a higher number of immunoreactive cells in the NTS and the 10N. These results may be interpreted as LPLRFamide causes anorectic effects in rats that are mediated by the brainstem. These results may aid in the development of anti‐eating treatments.
Metabolite‐detecting and adrenergic gene expression after 40K time trial performance during thermoneutral and heat‐stressed conditions
Authors: VanHaitsma Timothy A , Light Alan R , Light Kathleen C , Hughen Ronald W , White Andrea T
Published: June 2021

Strenuous exercise may be limited by metabolite detecting receptors on Group IV afferents which heat stress may exacerbate. This study compared mRNA quantities after prolonged intense exercise during thermoneutral (NT) and heat‐stress (HT) conditions on objective markers related to fatigue.

Ten cyclists (33.2 ± 3.0 yr, 57.2 ± 1.1 mL/kg/min) performed two 40k time trials; NT (21 C, 10% humidity (RH), temp change (CT) 1.93 C) and HT (35 C, 30% RH, CT 2.10 C). Blood was drawn at baseline, 0.5, 8, 24, and 48 hr after exercise. Leukocytes were separated for qPCR analysis of 5 metabolite detecting receptors (ASIC1, ASIC3, P2X4, TRPV1, TRPV4) and 3 adrenergic receptors (α2a, β1, and β2). Lactates were taken 1 and 5 min post‐ex.

Power and post‐ex lactates were lower and exercise time was longer for HT vs NT (p<.001). After both trials, decreases in ASIC3 and TRPV1 were seen (0.5 and 8 hr, p<.05), with larger decreases after NT (p<.05). Post‐ex ASIC3 and TRPV1 were related to 5 min post‐ex lactate (r=‐.50 and ‐.60, p<.01). ASIC1 decreased after NT (0.5, 8, and 48 hr, p<.05). P2X4 decreased after both trials (0.5 hr). TRPV4 decreased after NT (0.5 hr, p<.05) and HT (8 hr, p<.001). α2a increased at 0.5 hr after HT and β2 decreased 0.5 hr after NT.

Post‐ex decreases in metabolite detecting genes may represent downregulation of afferent signaling to reduce fatigue sensations. HT produced smaller changes in post‐ex ASIC1, ASIC3 and TRPV1 expression.
Intravenous ascorbic acid does not block the pressor or sympathetic nerve response to ischemic fatiguing rhythmic handgrip exercise
Authors: Muller Matthew David , Drew Rachel C , Cui Jian , Blaha Cheryl , Mast Jessica , Sinoway Lawrence I.
Published: June 2021

Reactive oxygen species stimulate group IV muscle afferents in rats but the effects of oxidative stress on the exercise pressor reflex in humans is unknown. The purpose of the current study was to determine the effect of intravenous ascorbic acid (Vitamin C) on the exercise pressor reflex in humans. Seven young healthy nonsmokers participated in the current study, which was part of a large protocol in our laboratory. On Day 1, they underwent systemic infusions of saline and then Vitamin C; on Day 2, they underwent a saline time/volume control. Muscle sympathetic nerve activity (MSNA), heart rate, and blood pressure were monitored throughout baseline, ischemic fatiguing rhythmic handgrip exercise, and post‐handgrip circulatory arrest. The change in mean arterial pressure from baseline to fatigue was not different between saline (Δ 35±6 mmHg) and Vitamin C (Δ 31±6 mmHg). Similarly, heart rate (Δ 22±4 bpm vs Δ 26±4 bpm) and MSNA burst rate (Δ 22±6 bpm vs Δ 22±5bpm) were also not different between treatments. Metaboreflex activation and time control experiments similarly showed no treatment effects. The current data suggest that oxidative stress does not contribute to the magnitude of blood pressure or MSNA response seen during fatiguing handgrip in young healthy subjects.

This study supported by P01 HL096570 (LIS) and UL1 RR033184 (LIS)
Blunted Arterial Baroreflex Sensitivity: A Contributor to Hypertension in Angiotensin Type 2 Receptor Knockout Mice
Authors: Gao Juan , Chao Jie , Walther Thomas , Gembardt Florian , Zucker Irving H. , Gao Lie
Published: June 2021

AT2R deficient (KO) mice have been reported to exhibit hypertension, which, we hypothesized, is related to the loss of sympathetic inhibition by the AT2R. In this experiment, we found that, compared to wild type (WT) mice: (1) KO mice exhibited higher blood pressure (MAP) (KO 85.0 ¡À 3.7 vs WT 74.7 ¡À 3.8 mmHg, p < 0.05) and renal sympathetic nerve activity (RSNA) (KO 31.3 ¡À 4.8 vs WT 18.5 ¡À 0.9 % max, p < 0.05); (2) Baroreflex sensitivity was blunted in KO mice (Gmax for HR: KO 2.7 ¡À 1.1 vs WT 8.4 ¡À 2.7 bpm/mmHg, p < 0.05; Gmax for RSNA: KO −1.2 ¡À 1.7 vs WT 12.9 ¡À 1.3 % max/mmHg, p < 0.05); (3) In KO mice, ICV Ang II evoked bigger pressor and sympatho‐excitatory responses, and ICV losartan (AT1R antagonist) induced a bigger fall of MAP and RSNA; (4) DHE staining showed a higher superoxide product in nucleus tractus solitarius (NTS) of KO mice; (5) Western blot analysis indicated higher AT1R, SOD1, nNOS, and eNOS protein expressions in the NTS of KO mice. These results demonstrate an impaired baroreflex function in AT2R deficient mice, which might evoke sympatho‐excitation and therefore hypertension. Our data also imply that impairment of baroreflex function in KO mice may be related to increased superoxide in the NTS via an upregulated AT1R expression and enhanced Ang II responses. The upregulated SOD1, nNOS, and eNOS protein expressions in NTS of AT2R KO mice may represent a compensatory mechanism to locally accumulated superoxide and deficient NO.
GABAergic neurons in the medullary raphé possess network independent chemosensitivity in situ
Authors: Iceman Kimberly E. , Richerson George B. , Harris Michael B.
Published: June 2021

The identity and location of central pH/CO2 sensitive chemoreceptors are not fully understood. Serotonin (5‐HT) and γ‐aminobutyric acid (GABA) synthesizing neurons in the medullary raphé have demonstrated intrinsic chemosensitivity in vitro. This evidence forms the basis for our “push‐pull” model of raphé contributions to chemosensitivity. We have previously shown that CO2‐stimulated 5‐HT neurons occur in the medullary raphé in situ. Here, we test the hypothesis that the medullary raphé contains GABA synthesizing CO2‐inhibited neurons that retain their chemosensitivity after pharmacological blockade of major fast synaptic inputs. To assess chemosensitivity, we record extracellular single neuron discharge during normocapnic and hypercapnic conditions within the medullary raphé of the unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation. Network dependence of chemosensitivity is assessed by application of antagonists for AMPA, NMDA, glycine, and GABAa receptors that disrupt fast‐synaptic network properties. Juxtacellular labeling and immunohistochemistry establish neurotransmitter phenotypes of recorded neurons. Results support independence of CO2‐inhibited GABA neuron chemosensitivity from fast synaptic inputs. Supported by NIH 54NS041069‐06A1.
Persistence of inspiratory rhythm in calibrated newborn rat pre‐Bötzinger complex slices upon blockade of store‐mediated calcium signaling
Authors: Ballanyi Klaus , Panaitescu Bogdan , Ruangkittisakul Araya
Published: June 2021

It is not clear which roles calcium release from cellular stores and subsequent modulation of ion channel activities play in generation of inspiratory rhythm by the bilaterally organized pre‐Bötzinger complex (preBötC) in the lower brainstem.

We studied this in transversal newborn rat brainstem slices with anatomically defined location of the <200 μm thin preBötC using suction electrode recording of inspiratory‐related neural population activity combined with contralateral multiphoton/confocal imaging of free cytosolic calcium (Cai). In 400 μm thick slices with centered or caudally exposed preBötC, inspiratory rhythm was not perturbed during 20 min bath‐application of the sarcoplasmic/endoplasmic reticulum calcium ATPase blocker cyclopiazonic acid (CPA, 50 μM) and remained unchanged for further >1 h after return to control solution. In 400 or 700 μm thick slices with caudally exposed preBötC, persistence of rhythm during 50 μM CPA was accompanied by a major Cai rise in presumptive astrocytes (soma diameter ~10 μm) imaged in slice depths of up to 100 μm. Contrary, CAP caused a minor increase in Cai baseline in larger (15–25 μm) neighboring cells that showed inspiratory‐related Cai rises, thus likely representing inspiratory preBötC neurons. CPA attenuated astrocytic Cai rises due to glutamate (0.5 mM) and blocked (oscillatory) Cai rises in response to the metabotropic glutamate receptor agonist t‐ACPD (25 μM), whereas a major and modest response of preBötC neurons to glutamate or t‐ACPD, respectively, persisted in CPA.

Our findings indicate that CPA causes store‐mediated calcium release in astrocytes and impairs their metabotropic calcium signaling, whereas inspiratory bursting of preBötC neurons is independent of such calcium stores.

Supported by CIHR, AHFMR, CFI‐ISRIP, CIHR‐MFN Health
Oxidative stress in the respiratory response to intermittent hypoxia in athletes and sleep apnea patients: similar mechanisms but opposite outcomes
Authors: Pialoux Vincent
Published: June 2021

The overproduction of reactive oxygen species (ROS) observed in response to intermittent hypoxia may either trigger adaptive physiological processes or play a role in mechanisms leading pathological situation such as respiratory disturbance. In a series of articles, we report that this ROS increase is likely involved in beneficial or detrimental adaptations depending to the intensity of the hypoxic stimulus and on the health state of the subject. In this context, oxidative stress appears to play a role in the signalling process that controls the hypoxia inducible factor 1 (HIF‐1). Although ROS is needed for stabilization of HIF‐1α, we demonstrated in humans submitted to moderate hypoxia, that ROS may also be involved in the increase of HIF‐1α transcription. Then, we observed in athletes that the ventilatory acclimatization to hypoxia after intermittent altitude training was improved by elevated oxidative stress through a stimulation of HIF‐1α pathway. Conversely, by exposing healthy subjects to chronic intermittent hypoxia simulating obstructive sleep apnoea (OSA), we reported that oxidative stress increased hypoxic ventilatory responses to hypoxia. This increase could promote unstable breathing and thereby might perpetuate OSA as it was previously suggested. Founded by International Olympic Committee, Alberta Heritage Foundation for Medical Research and Canadian Institutes of Health Research
Astrocytes in NTS after exposure to chronic intermittent hypoxia
Authors: Yamamoto Kenta , Yuan Chengbo , Eubank Wendy , Franzke Michelle , Mifflin Steve
Published: June 2021

Chronic intermittent hypoxia (CIH) is a model of the arterial hypoxemia that accompanies sleep apnea and increases resting arterial pressure. Exposures to hypoxia activate astrocytes in the brain. Astrocytes are a primary source of brain angiotensinogen, the precursor to angiotensin II. We tested the hypothesis that chronic intermittent hypoxia increases astrocytes in the nucleus tractus solitarii (NTS). Sprague‐Dawley rats (13 ± 0.3 weeks old) were exposed to CIH (9% oxygen for 3min every 10 min, 8 h per day) for 7 days (n = 5) while control rats (n = 4) were maintained in normoxia. After the CIH protocol, brain stem tissue containing NTS was obtained. Protein levels of the astrocyte marker glial fibrillary acidic protein (GFAP) in the NTS were assessed by Western blot analysis. Relative protein levels of GFAP to actin in the caudal NTS did not differ between CIH and control groups (3.3 ± 1.7 vs. 4.0 ± 1.7, CIH vs. control, P=0.59). The GFAP levels tended to be lower in CIH than in the control group in intermediate and rostral NTS (intermediate: 2.3 ± 1.1 vs. 3.7 ± 0.6, P=0.11; rostral: 1.5 ± 0.9 vs. 2.7 ± 1.0, P=0.052). These results did not support the idea that hypertension induced by CIH is associated with an activation of astrocytes in the NTS. CIH might decrease astrocytes in the NTS as opposed to our hypothesis. Supported by HL‐088052
Changes in reactive oxygen species (ROS) in central respiratory centers with sustained hypoxia
Authors: Popa Daniel , Go Ariel , Fu Zhenxing , Powell Frank
Published: June 2021

ROS are important for plasticity in chemoreceptors and respiratory motor neurons with acute and chronic intermittent hypoxia. Recent studies show ROS change with sustained hypoxia so we hypothesized ROS contribute to plasticity with chronic sustained hypoxia (CSH) in ventilatory acclimatization to hypoxia. To test this, we measured (1) ventilation (Vi) in conscious, unrestrained mice breathing 21% or 10% O2 using barometric pressure plethysmography, and (2) ROS levels in medullary respiratory centers by imaging dihydroethidium (DHE) fluorescence in fixed tissue sections obtained after 2 hr of DHE exposure in 4 groups of mice (n=3–5 each): normoxic control (CON) and CSH (PiO2 = 70 Torr, 7 days), treated with a superoxide scavenger (MnTMPyP 5 mg/kg ip) or saline for 10–13 days prior to study. DHE fluorescence was observed in neurons but not glia. CSH decreased ROS to 60–71% of CON in nucleus tractus solitarii and hypoglossal neurons (NTS and XII). MnTMPyP decreased ROS in NTS and XII by 37% in CON but only 6–12% in CSH. In CON, Vi increased with MnTMPyP. Vi increased with CSH as expected but was not changed by MnTMPyP. The results are consistent with ventilatory inhibition by ROS that are scavenged by MnTMPyP and decreased in CSH. Supported by NIH RO1 HL‐081823 and 1P01 HL 098053.
Neuropeptide Y Signaling in Altered Catecholamine Synthesis during Intermittent Hypoxia
Authors: Kumar Ganesh K , Raghuraman Gayatri , Prabhakar Nanduri R
Published: June 2021

Intermittent hypoxia (IH) increases catecholamine (CA) levels in the adrenal medulla. In this study we examined whether the IH‐induced increase in CA is due to alterations in tyrosine hydroxylase (TH), the rate‐limiting enzyme in CA biosynthesis. Rats and mice were exposed to IH (15s of 5% O2; 5 min of 21% O2; 8 h per day for 10 days) or normoxia (21% O2) and adrenal medullae were harvested for analyses. IH increased TH activity and TH phosphorylation at ser‐40 (~ 3 fold), without altering TH protein levels. In addition to CA, adrenal medulla also expresses neuropeptide Y (NPY) which is known to modulate TH activity via activation of G‐protein coupled Y1 receptors (Y1R). IH markedly up regulated the expression of NPY levels, Y1R expression, cAMP levels and protein kinase A (PKA) activity. The contribution of NPY in IH‐induced TH activation was examined using 4‐phenyl‐3‐butenoic acid (PBA), an inhibitor of bioactive NPY synthesis. PBA treatment prevented IH‐induced increases in NPY levels, TH activity, TH phosphorylation (ser‐40) and CA levels. Furthermore IH‐induced increases in TH phosphorylation and activity and up regulation of PKA activity were absent in Y1R−/−but not in Y2R−/− mice. Taken together, these results suggest that NPY‐Y1R‐PKA signaling mediates IH‐induced increase in CA synthesis in the adrenal medulla (supported by grants from NIH HL‐89616 and HL‐90554).
Brainstem areas activated by intermittent apnea in awake unrestrained rats
Authors: Schoorlemmer Guus H. , Falquetto Barbara , Ferreira Caroline B. , Takakura Ana C. , Moreira Thiago S. , Cravo Sergio L.
Published: June 2021

Patients with obstructive sleep apnoea experience repeated hypoxia, hypercapnia, and changes in pulmonary afferent traffic, resulting in autonomic activation and hypertension. We analyzed brain areas activated by apnea in conscious rats by c‐fos immunohistochemistry. Wistar rats (n= 9, 300–380 g) were anaesthetized with ketamine + xylazine, and balloon‐tipped catheters were implanted in the trachea. The balloon tip was contained in a rigid thin‐walled Teflon tube, 6 mm long, 2.2 mm diameter. Inflation of the balloon closed the lumen of the Teflon tube, and induced obstructive apnea without inducing pain in the trachea. The free end of the catheter was exteriorized on the rat's back. One week after surgery, five rats were subjected to intermittent apnea (14 s each 2 min) for 1 h, and sacrificed 30 min after the end of apneas for analysis of c‐fos. The four remaining rats were used as controls. Apnea increased the number of c‐fos immunoreactive cells in the parabrachial and Kölliker‐Fuse complex, retrotrapezoid nucleus, commissural and dorsomedial nucleus of the solitary tract and both the rostral (RVLM) and caudal ventrolateral medulla (CVLM). Tyrosine hydroxylase was present in many of the c‐fos positive neurons in the C1 region (69 ± 3%), but not in the A1 or A2 catecholaminergic regions. In conclusion, apnea activates specific brainstem areas involved in autonomic control.

Financial support: FAPESP
Corticotropin‐releasing factor involves in promotive effect of restraint on neuronal activation by methamphetamine
Authors: Yuan Zung , Lin Yan-Wei
Published: June 2021

The mesocorticolimbic (MCL) system is the main place methamphetamine (MA) manifests its addictive characteristics. Stress is one of the risk factors engaging in initiation, recurrence and relapse of drug‐taking behaviors, but the mechanism is still ambiguous. Corticotropin‐releasing factor (CRF) acts as an integrator of stress responses. So, we planned to clarify the role of CRF on the promotive effect of restraint (RST) on MA‐induced neuronal activation in nuclei of the MCL system by immunohistochemistry. Either RST (30 min) or MA (2.5 mg/Kg, ip) induced increase the number of cFos‐immunoreactive (‐ir) cells in nucleus accumbens (NA) and medial prefrontal cortex (mPFC). RST prior to MA challenge induced more neuronal activation. The promotive effect of RST was eliminated by central injection of CRF receptor antagonist, alpha‐CRF9‐41. So, we proposed that RST may potentiate stimulatory effect of MA by activating CRF signaling. For tracing the origin of CRF signal, retrograde tracer, fluro‐gold, was injected in the NA. Besides ventral tegmental area (VTA) and substantia nigra, lots of neuronal were labled with FG in the basolateral amygdale which receives CRF‐ir innervation. So, the stress signal may be transmitted into the MCL system through BLA by CRF signaling. This work was sponsored by Tzu Chi Foundation
Integration of the baroreflex control mechanism in a heterogeneous model of the cardiovascular system
Authors: Fernandes Luciano Gonçalves , Trenhago Paulo Roberto , Blanco Pablo Javier , Feijóo Raúl A
Published: June 2021

The present work describes the mathematical model of the baroreflex receptor mechanism working on a dimensionally‐heterogeneous (1D‐0D) closed‐loop model of the cardiovascular system. Such heterogeneous model includes the 1D description of the arterial tree, 0D model for the venous, pulmonary and cardiac circulation. The detailed description of the arterial vascular tree allow us to perform vasomotor control actions in a different way, while gaining insight about the effects of the baroreflex regulation over hemodynamic quantities of interest throughout the entire vascular system. One example is presented: we simulate the hemorrhage in the abdominal aorta artery and analyze the action of the baroreflex over the system.

CAPES, CNPq and FAPERJ
Accelerated Bone Healing in the Absence of TLR4
Authors: Wang Dan , Kubala Adam , Billiar Timothy , Cooper Greg
Published: June 2021

OBJECTIVE

Toll‐like receptor 4 (TLR4) is a member of highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate both the immune response and bone metabolism during long‐bone fracture healing. Unlike defects in long bones, defects in the calvaria often do not heal. Here we tested the hypothesis that the inflammation driven by TLR4 activation limits the healing of calvarial defects.

METHODS

Circular bone defects were made in the parietal bones using a 1.8mm outer diameter trephine in WT (n=32) and TLR4−/− mice (n=24). Calvarial bone and surrounding soft tissues (e.g. skin and brain) were harvested and bone healing was assessed using histomorphology at postoperative days 0, 1, 2, 4, 7, 14, 21 and 28.

RESULTS

Histomorphometric analysis demonstrated significantly larger bone healing areas in TLR4−/− mice compared to WT mice on day7 (p<0.001). Similar levels of bone healing were observed by day 28 (p=0.070).

CONCLUSIONS

These data are consistent with the hypothesis that TLR4 activation delays calvarial healing in mice. Further work is required to determine if this is due to inflammation driven by TLR4 activation.
Six3 expression is augmented in the facial primordia of mice with misexpression of six2 associated with frontonasal dysplasia
Authors: Hynd Thomas , Fogelgren Ben , Somponpun S. Jack , Fong Sheri F. T. , Lozanoff Scott
Published: June 2021

We have previously described the Br mutant mouse displaying heritable frontonasal dysplasia. Linkage analysis mapped the mutation near the homeobox transcription factor six2, normally expressed in the facial mesenchyme during development. The purpose of this study is to determine expression patterns of six2, as well as possible upstream and downstream targets of six2, in the developing midface. The three sets of facial prominences (medial, lateral, and maxillary) of E11.5 embryos were dissected and RNA extracted for qPCR assays and microarray analysis. Medial nasal prominences (MNP) were also taken for cell culture. qPCR results indicated six2 expression is highest in the MNP and demonstrated haploinsufficient down‐regulation in each of the three facial prominence sets in the Br mouse. Microarray results suggested the misregulation of several genes in the Br midface, including another member of the Six family of transcription factors: six3. qPCR and immunohistochemistry for six3 substantiated its upregulation in the microarray. Future work using RNA interference on six2 will be used to investigate possible pathways involving six2 and six3. Preliminary results using an in vitro knockdown of six2, performed on an MNP cell culture system utilizing siRNA, demonstrated a 65% knockdown of six2. These results may enable further in vitro work in order to elucidate a pathway in the developing midface involving six2.

Grant Funding Source : NIH R01DK064752 (SL) & NCRR 5P20RR024206 (SJS)
A Novel Gene Crispld2 may Contribute to Facial Dysmophology in a Chicken Model of Crouzon's Syndrome
Authors: Li Xin , Tropp Stephen , Young Nathan , Hallgrimsson Benedikt , Marcucio Ralph
Published: June 2021

Craniosynostosis causes malformations of the skull that are characterized by premature suture closure, as well as malformations of the brain and face. Recent work in mice, and our data, suggest that the malformations in the brain and face are a primary consequence of mutations that produce craniosynostosis. Mutations in FgfR1–3 account for ~25% of craniosynostosis syndromes. To investigate mechanisms underlying these facial malformations, we infected chick embryos with a retrovirus encoding FgfR2C278F. Compared to controls, treated embryos have widened‐faces and midfacial dysplasia resembling defects seen in craniosynostosis. Further, microarray analysis detected changes in gene expression. In particular, Crispld2 (cysteine rich secretory LCCL domain protein 2), a nonsyndromic cleft lip/palate related gene, is downregulated. The function of Crispld2, and how it may contribute to cleft lip, is unknown. We detected Crispld2 expression in facial mesenchyme in chicken embryos at HH 22. To assess function of this gene, we created an RCAS vector encoding Crispld2. Infected chicken fibroblasts appeared to migrate faster than controls cells, but embryos infected with the virus appeared normal. Therefore, we are currently investigating the effect of knocking‐down Crispld2. In conclusion, we have identified a novel gene Crispld2 and are determining the role this gene plays in craniofacial development.

Grant Funding Source: NIH
Anatomy of the Incomplete Ureteral Duplication
Authors: Mehta Mudresh , Bui Trang , Rengasamy Padmanabhan
Published: June 2021

Ureteral duplications constitute the most common congenital anomalies of the urinary tract. Incomplete duplications are associated with uretero‐uremic reflex or uretero‐pelvic junction obstruction. Here we report an incomplete duplication of the left ureter observed in a 75 year old male cadaver. The medial ureter emerged from the upper end of the hilum, passed posterior to renal, gonadal and left colic vessels and fused with the lateral ureter halfway on the lateral pelvic wall. The lateral ureter emerged from the lower hilum, crossed the psoas, approached the medial one at the left common iliac artery level, both crossed the internal iliac artery, united with each other, crossed the vas, turned medially and dumped into the bladder. Throughout their course, the ureters were retroperitoneal. The common ureter was almost twice as thick as the duplicated segments. Duplicated segments were accompanied by a rich vascular network. A Y‐shaped vein with one arm coming from the renal vein and the other from the lower pole of the kidney connected to the left gonadal vein. The left colic vein dumped into the left gonadal vein. There were two testicular arteries ‐ one from the renal and the other from the aorta. Renal artery divided precociously adjacent to the aorta and behind the renal vein. The right kidney and ureter were normal. Right gonadal vein dumped into right renal close to its union with the inferior vena cava. The cause of anomalous vascular connection of the left gonadal vein associated with ureteral duplication is not known.

Grant Funding Source : Mercer University
Collagen analysis of mesenchymal stromal cell assisted hernial scar repair
Authors: Krontiris-Litowitz Johanna , Dorion Heath , Fagan Diane L. , Ferrari Jonathan , Holmes Jonathan , Marie Hazel , Heffner Jeremy
Published: June 2021

Mesenchymal stem cells (MSC) migrate to the site of injury to promote tissue remodeling and cellular re‐organization. In this study we use a hernia repair model in rats to investigate how MSCs and platelet rich plasma (PRP) influence collagen deposition in hernial scars. A midline incision in 21 age‐matched Lewis rats was repaired using one of 3 protocols: 1) standard suture repair (control) 2) platelet‐rich plasma embedded in a collagen matrix (PRP) and 3) platelet‐rich plasma and mesenchymal stem cells embedded in a collagen matrix (MSC+PRP). At 4 weeks post surgery, 0.15 mm scar tissue was harvested and analyzed for collagen content and phenotype. Results showed that collagen content was greater in MSC‐treated rats. Total collagen in MSC+PRP scars was 394.2 % greater than controls (p=0.003) and 250.4% greater than PRP scars (p=0.004). Collagen concentration (per mg scar protein) was significantly higher in MSC+PRP scars than in controls (p=.012) and there was no significant difference between control and PRP scars. A comparison of collagen I/III ratios in scar tissue using 2Dgel analysis of CnBr treated scar tissue indicated that the relative proportion of Col III was higher in MSC+PRP treated scars than in control. The results of this study show that MSC therapy can enhance hernial scar integrity by increasing collagen content and decreasing the Col I/III ratio. Supported by the American Hernia Society.
Vesicant‐Induced Autophagy
Authors: Gordon Marion K , Raman Lakshmi , Hahn Rita A , Schlager John J , Babin Michael C , Gerecke Donald R , Svoboda Kathy K H
Published: June 2021

Autophagy clears aggregating proteins in some neurodegenerative diseases (Ravikumar et al, 2002; Webb et al., 2003). Sulfur Mustard (SM) alkylates and crosslinks intracellular and extracellular proteins, lipids and DNA. We hypothesized that autophagy is induced by SM‐exposure to degrade alkylated products. Rabbit eyes were exposed to 0.4 microL SM applied directly onto the cornea. Sacrifice was at 1, 3, 7, and 28 days post‐SM. Corneas were collected, and autophagosomes were counted in electron micrographs. Epithelia (N=100) and stromal keratocytes (N=50) of unexposed and exposed corneas were analyzed at each time point. Autophagosomes were 7 times more abundant in the epithelium, and 5 times more abundant in stromal cells at 3 and 7 days post‐SM, compared to unexposed corneas. By 28 days post‐SM, the number was 3–4 times higher in both cell types than in unexposed controls. Thus, the highest level of autophagy occured between 3 and 7 days post‐SM, and declined by 28 days, but was still 4 times higher than unexposed controls. The results of this analysis suggested that autophagy is one mechanism used by SM‐exposed corneas to degrade alkylated proteins.
Hepatocyte maturation is arrested in mouse liver lacking the splicing factor SRSF3 (SRp20)
Authors: Sen Supriya , Webster Nicholas JG
Published: June 2021

SRSF3 is the smallest member of the highly conserved SR protein family that has numerous roles in mRNA metabolism, from transcription to translation. To investigate the role of SRSF3 in postnatal hepatic function, we used Cre‐loxP‐mediated gene technology to delete SRSF3 specifically in hepatocytes in mice. Deletion of SRSF3 inhibits hepatocyte maturation as livers from mice lacking SRSF3 exhibit a fetal phenotype characterized by increased expression of the fetal isozyme of pyruvate kinase (M‐PK), persistent expression of alpha‐fetoprotein and H19 mRNA. The livers also show increased expression of the fetal isoform of the insulin receptor (IR‐A). The expression of several genes that regulate glucose and lipid metabolism in the mature liver is significantly reduced in SRSF3−/− liver, and glycogen levels are decreased. Global gene expression and exon microarray analyses identified multiple splicing and expression changes in the SRSF3−/− liver. Interestingly, some of these changes alter expression of major hepatic transcription factors that play crucial role in development and metabolism. Thus, SRSF3 directs a genetic program required for morphological and functional differentiation of hepatocytes and the hepatocyte SRSF3 knock‐out mice provide a new model to study metabolic dysregulation and liver disease.

This work has been supported by VA Merit Grant Award.
Native Accessible ChIP: A Novel Approach to Interrogate Protein‐DNA Interactions in Accessible Chromatin
Authors: Wang Yan , Meng Xiangdong , Kong Michelle , Okino Steven T
Published: June 2021

We have developed a novel technology for isolating accessible chromatin. When cells are permeabilized and treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin can be easily isolated and characterized. We now report that the isolated accessible chromatin is still associated with its cognate DNA‐binding proteins and histones. Based on this, we developed a novel chromatin immunoprecipitation (ChIP) procedure that does not require protein‐DNA cross‐linking or DNA shearing. Using this method, termed NA‐ChIP (for native accessible ChIP), we demonstrate that RNA polymerase II (RNAPII), TBP and H3K4me3 are all associated with active promoters, but are rarely detected at silenced promoters. Comparison of NA‐ChIP and a standard ChIP assay shows that immunoprecipitation (IP) efficiency is similar, but IP specificity is dramatically different. To measure IP specificity we compared the ratio of active promoter DNA precipitated with RNAPII (signal) relative to inactive promoter DNA precipitated (noise). We find that NA‐ChIP is highly specific with a signal‐to‐noise ratio >500; standard ChIP is less specific with a signal‐to‐noise ratio of 9. We conclude that NA‐ChIP offers significant advantages over standard ChIP, including dramatically reduced background and an easier workflow.
Structural studies of the histone chaperone Hif1, a component of the Hat1 chromatin assembly complex
Authors: Bigeh Allison , Pickle Elyssa , Bagley Michael J. , Seangmany Nessa , Dutnall Robert N.
Published: June 2021

The Hat1 chromatin assembly complex contains the Hat1 histone acetyltransferase and two Hat1‐interacting factors: Hat2 and Hif1. The Hat1 enzyme specifically acetylates histone H4 while Hat2 and Hif1 are both histone binding proteins. These three proteins form cytoplasmic and nuclear complexes that are part of a pathway that deposits newly synthesized histone H3 and histone H4 onto DNA. In yeast, Hat1 complexes contribute to gene regulation and DNA repair. Hat1, Hat2 and Hif1 are each highly conserved and form similar complexes in organisms ranging from yeast to humans. We are using biochemical and biophysical methods to study the structure and function of the Hif1 protein from yeast with the goal of understanding how it contributes to the activity of the Hat1 chromatin assembly complex. Hif1 has histone chaperone activity and contains four tetratricopeptide (TPR) sequence motifs. We have evidence that like its human counterpart, bacterially expressed yeast Hif1 is an oligomer forming dimers and possibly tetrameric assemblies. The bacterially expressed protein also interacts with histones. We are currently using truncation mutants to map the regions of the Hif1 protein involved in oligomer formation and histone binding. We are also using a variety of biochemical and biophysical methods to probe the strength and specificity of Hif1‐histone interactions.
Role of the TWIST proteins and ADD1 in the regulation of the CHRDL1 gene
Authors: Casasnovas Jose , Franco Hector Luis , Cadilla Carmen Lydia
Published: June 2021

Puerto Rican Setleis Syndrome (SS) patients harbor a nonsense mutation (TWIST2‐Q119X) that causes truncation of the C‐terminal region required for transactivation of target genes, resulting in facial and skin abnormalities. We found that the chordin‐like 1 (CHRDL1) gene is up‐regulated in SS patients and silencing of TWIST2 causes an increase in CHRDL1 gene expression. CHRDL1 codes for a bone morphogenic protein antagonist expressed in mesenchymal and neural tissues. Putative TWIST binding sites were found at positions −2421, −2051, −1000, −763, and +46 relative to the TSS of the CHRDL1 gene. EMSA analysis showed specific binding activity of TWIST1 and TWIST2 homodimers to the −2421 putative site, which TWIST2‐Q119X could not bind. An adjoining site was bound by SREBP1c/ADD1, a transcription activator inactivated by TWIST2. EMSA analysis suggests that TWIST2 and ADD1 compete for binding the same − 2421 site. Luciferase reporter assays revealed that the 3000bp CHRDL1 gene upstream region drives its expression and ADD1 increases it 2.6X over basal levels. TWIST2, but not Q119X, was able to block activation by ADD1, while overexpression of TWIST2 Q119X results in increased expression. We suggest that the TWIST2 Q119X mutant protein cannot bind to the −2421 site nor compete with ADD1, but may sequester other transcription factors important for CHRDL1 repression. Supported by NIH Grants G12RR03051 and R25GM061838
Mutagenisis of Argonaute 2 in studying microRNA loading
Authors: Doxtader Katelyn Ashley , Androsavich John , Walter Nils
Published: June 2021

MicroRNAs (miRNA) are small, non‐coding RNAs that play a critical role in post‐transcriptional gene regulation by acting as a guide for directing the repressor protein Argonaute 2 (Ago2) and its RNA‐induced silencing complex (RISC) to mRNA targets. Recently, we reported on the dynamic movements and localizations of individual miRNAs in living cells, thus demonstrating the feasibility and potential of using single molecule analysis to study non‐coding RNAs in the cellular environment. Our goal is to now extend these studies to the Ago2 protein, the central effecter of the miRNA pathway. Rudell et al (2011) recently found that mutation of the conserved tyrosine Y529 located in the 5′‐end‐binding pocket of Ago2 to a negatively charged glutamate mimics phosphorylation by an as‐yet‐unknown kinase and inhibits small RNA binding. We plan to incorporate this mutation using site‐directed mutagenesis into a plasmid encoding a human Ago2‐EGFP fusion. Using fluorescence microscopy, we will then monitor and compare the localizations and diffusive properties of EGFP‐Ago2 Y529E to its wild‐type counterpart in HeLa cell lines under transient expression. Since EGFP‐Ago2 Y529E is incapable of binding miRNA, it will enable us to distinguish the diffusive motions of miRNA‐loaded RISC from unloaded‐RISC, and to characterize and potentially quantify miRNA loading, a critical step in the miRNA pathway.
Preliminary in vitro functional analysis of the DEAD‐box protein DDX3
Authors: Floor Stephen N , Smith Alison , Zhou Kaihong , Doudna Jennifer A
Published: June 2021

DEAD‐box proteins are integral to RNA processing in cells, including the transcription, splicing, export, and translation of messenger RNA, assembly of ribosomes, and chaperoning of RNA folding. The DEAD‐box protein DDX3 (yeast Ded1) is implicated in facilitating transcription, splicing, nuclear export, and translational control, and is additionally thought to potentiate interferon production resulting from cytoplasmic pattern recognition receptor signaling. DDX3 is also hijacked by pathogens including HIV and HCV, transforms breast cells in culture, and is dysregulated in hepatocellular carcinoma. However, little is known regarding the precise mechanism of DDX3 in any of these processes. The aim of this work is to illuminate DDX3 function and regulation using structural and biochemical methods. We present preliminary in vitro reconstitution experiments determining the association of DDX3 with protein cofactors and their functional implications. This work builds a foundation to examine the function of DDX3.
Analysis of Small RNAs Associated with Plant Senescence
Authors: Wright Christopher D , Thatcher Shawn R , Burd Shaul , Lers Amnon , Green Pamela J
Published: June 2021

Since their discovery in 1993, small RNAs have been shown to have an increasingly important role in post‐transcriptional gene regulation. MicroRNAs (miRNAs) are of particular interest because they are often conserved across many different species. This allows research on model organisms to be more easily extended to help solve problems in commercial sectors. One major problem in the agricultural sector is the decline in health during plant aging, or senescence. This process is of key importance commercially, where it can cause decreases in crop yield and postharvest quality. If the onset of senescence could be delayed or halted, based on knowledge of the mechanisms involved, this could prove very beneficial for agricultural improvement. To test the hypothesis that miRNAs participate in senescence, we constructed and sequenced a series of small RNA libraries from Arabidopsis leaf tissue at varying stages of senescence. A comparison of miRNA expression changes in these libraries provided evidence for novel senescence regulation. Using northern blots as a confirmation, we have reproduced the regulation of previously published senescence‐dependent miRNAs, as well as identified several new candidates. We have also constructed Parallel Analysis of RNA Ends (PARE) libraries to search for mRNA cleavage products due to specific miRNA‐guided cleavage, as well as discover general senescence‐related RNA decay patterns. Initial analysis of PARE data has revealed regulated cleavage of a target of one of the senescence‐regulated miRNAs at the expected site during senescence. Further analysis and validation of both small RNA as well as PARE library data will help us gain a deeper understanding of the fundamental processes involved in senescence. Supported by BARD, NIH, and HHMI.