bluuebunny/crossref_metadata_2025 huggingface dataset:
Time Lapse Measurement of Mitochondrial Membrane Potential in Absolute Millivolts in Single Intact Cells
Authors: Gerencser Akos A , Chinopoulos Christos , Birket Matthew J , Jastroch Martin , Vitelli Cathy , Nicholls David G , Brand Martin D
Published: June 2021

Assaying mitochondrial membrane potential (ΔΨM ) in absolute millivolts in intact cells had been previously limited mostly to radioisotope distribution methods. Here we introduce a purely fluorescence based ΔΨM assay to calculate time courses of ΔΨM in absolute millivolts in monolayer cell cultures. We built a biophysical model‐based method to calibrate single‐cell fluorescence of a bis‐oxonol‐type plasma membrane potential (ΔΨP ) indicator and the ΔΨM –probe TMRM to potentials. The ΔΨP ‐dependent distribution of the probes is modeled by Eyring rate theory, which we have verified using fluorescence imaging combined with voltage clamp. ΔΨM is determined in millivolts by deconvoluting TMRM fluorescence in time taking in account the slow, ΔΨP ‐dependent redistribution and its Nernstian behavior. The resting ΔΨM is calculated from a complete step‐depolarization of ΔΨM. The calibration accounts for volume ratios, high and low affinity bindings, activity coefficients, background fluorescence and optical dilution. All of the calibration parameters are back‐calculated from fluorescence intensities or measured by confocal microscopic assays (validated by electron microscopy), allowing comparisons of potentials in cells with different properties. We show that ΔΨM in cultured rat cortical neurons is regulated between −120 and −154 mV as a concerted effect of increased ATP turnover and Ca2+‐dependent metabolic activation.
Docosahexaenoic acid (DHA) Alters Mitochondrial Membrane Fluidity and Attenuates ROS induced Calcium Release in Hypertrophied Myocardium
Authors: Dabkowski Erinne , O'Connell Kelly , Ribeiro Rogerio , Xu Wenhong , Galvao Tatiana , Stanley William
Published: June 2021

Treatment with ù‐3 PUFAs high in DHA prevents cardiac dysfunction and improves mitochondrial function in rat models of heart failure. Mechanisms responsible for the effects of DHA on mitochondria are not well known. The goal of this study was to determine the effects of DHA supplementation on mitochondria in hypertrophied myocardium. Hypertrophy was induced by transverse aortic constriction (TAC) in rats. Four days following surgery, animals were placed on either standard or high DHA diet. At 14 weeks post surgery, echo was performed, animals were sacrificed and mitochondria isolated. Echo revealed significant decreases in ejection fraction (EF) and fractional shortening (FS) in TAC vs. Sham groups that was significantly attenuated with DHA. Analysis of mitochondrial hydrogen peroxide showed increases in ROS from complex III in standard TAC vs. Sham which was attenuated with DHA. Infusion of hydrogen peroxide resulted in a higher rate of calcium release from the mitochondria in both TAC vs. Sham groups. Calcium release was delayed in DHA TAC as compared to STD TAC. There were no changes in mitochondrial membrane fluidity between standard TAC and Sham animals. However, there was an increase in fluidity between DHA TAC and all other groups. These data suggest that enhanced mitochondrial membrane fluidity and attenuation of ROS production with DHA may underlie improved cardiac function in hypertrophied myocardium.
Hypertension and Pregnancy are accompanied by differential changes in GABAA and NMDA receptor subunit expression in the hypothalamic Paraventricular Nucleus
Authors: Cork Simon , Chazot Paul , Pyner Susan
Published: June 2021

Sympathoexcitation is characteristic of cardiovascular disease and pregnancy. Sympathoexcitation emanates, in part, from the hypothalamic paraventricular nucleus (PVN). Hypertensive rats show that the normal balance of GABAergic inhibition and glutamatergic excitation is altered. We hypothesise that this alteration is caused by changes in the subunit composition of the GABAA and NMDA receptors in the PVN. Brains from female Wistars, female spontaneously hypertensive rats (SHR) and pregnant Wistar rats (E19) were subjected to either immunoblotting of PVN micropunches or immunohistochemistry for NMDA and GABAA receptor subunits. Expression of NMDA subunits GluN1, ‐2A and ‐2B was found in all subnuclei of the PVN. A significant increase in expression of the GluN2A subunit was observed in the PVN of SHR (P<0.05 n=3). Furthermore, a significant reduction in GABAA α1 was observed in the PVN of SHR and pregnant animals (P<0.05, n=3). A significant reduction in expression of the GABAA α5 subunits was observed in the PVN of SHR animals (P<0.001 n=3).

We hypothesise that the sympathoexcitation observed in pregnancy is driven by a withdrawal in phasic GABAergic inhibition. Conversely, we believe that in SHR, the sympathoexcitation is driven by a decrease in both phasic (α1) and tonic (α5) GABAA‐mediated inhibition, as well as an increase in NMDA‐driven excitation.

Work supported by the BBSRC
Metabolic and cardiovascular adjustments to hemorrhage in [TGR(ASrAogen)] rats and (mRen2)27 rats
Authors: de Lima Daniel C , Shaltout Hossam A. , Coimbra Candido Celso , Diz Debra I
Published: June 2021

We assessed the metabolic and cardiovascular adjustments to hemorrhage in rats with low brain angiotensinogen [TGR(ASrAogen)] (Aogen) versus high brain renin [(mRen2)27] (mRen27) rats. Sprague Dawley rats (SD) were used as a control group. Conscious mean arterial pressure (MAP), heart rate and plasma glucose were assessed before and during the 30 minutes after hemorrhage (1.2 mL/100g body weight/2min) in rats previously instrumented with jugular and femoral artery catheters. The resting MAP was lower in ASrAogen and higher in (mRen2)27 relative to SD rats [SD, 104 + 5 mmHg; Aogen, 74 + 3 mmHg; (mRen)27, 149 + 13 mmHg]. In comparison to SD, the hemorrhagic hypotension was less in ASrAogen rats (vs SD, 37%, p<0.05) and double in (mRen2)27 rats (vs ASrAogen, p<0.05). While the heart rate did not change in ASrAogen during hemorrhage, SD and (mRen2)27 rats had pronounced bradycardia (P<0.05). The hemorrhagic hyperglycemia response was stronger in ASrAogen) rats compared to SD (63%; p<0.05), whereas the metabolic response in (mRen2)27 rats was not different from control rats. In summary, ASrAogen rats with a targeted deficit in glial angiotensinogen presented a greater hyperglycemia response and less hypotension to hemorrhage, suggesting they are better at mounting the sympathetic nervous system activation necessary for hemodynamic stability in response to acute hemorrhage than either(mRen2)27 or SD rats. CNPq; NIH
Sex differences in sympathetic neural responses to 24‐hour sleep deprivation in humans
Authors: Yang Huan , Durocher John J , Larson Robert A , DellaValla Joseph P , Carter Jason R
Published: June 2021

Sleep deprivation has been linked to hypertension, and recent evidence suggests that associations between sleep deprivation and hypertension are stronger in women. We hypothesized that 24‐hour total sleep deprivation (TSD) would elicit augmented pressor and sympathetic neural responses in women compared to men. Resting heart rate (HR), blood pressure (BP), and muscle sympathetic nerve activity (MSNA) were measured in 30 healthy subjects (age, 22±1 yr; 15 men and 15 women). Relations between diastolic BP and MSNA were used to assess sympathetic baroreflex function. Subjects were studied twice, once after normal sleep and once after TSD (randomized, crossover design). TSD elicited similar increases in BP in men and women. TSD reduced MSNA in men (25±2 to 16±3 bursts/100 heart beats; P=0.02), but not women. TSD did not alter sympathetic baroreflex sensitivity in either sex, but the operating point was shifted downward and rightward in men (40±4 to 27±7 units; P=0.03). TSD reduced testosterone in men, and these changes were correlated to changes in resting MSNA (r=0.59, p=0.04). Resting HR and estradiol were not altered by TSD in either sex. In conclusion, TSD‐induced hypertension occurs in both sexes, but only men demonstrate altered resting MSNA. The sex differences in MSNA are associated with sex differences in sympathetic baroreflex function (i.e., operating point) and testosterone. Supported by NIH (HL‐ 098676).
Hydrogen sulfide contributes to the enhanced chemoreflex ventilatory response to acute hypoxia in heart failure rats
Authors: Del Rio Rodrigo , Marcus Noah J , Stein Gregory , Schultz Harold D
Published: June 2021

Increased sympathetic outflow is a major contributor to the progression of heart failure (HF). Potentiation of the peripheral chemoreflex has been related to the activation of the sympathetic nervous system in HF patients and in animal models. The carotid body (CB) plays a pivotal role in the increased peripheral chemosensitivity in HF animals. Hydrogen sulfide (H2S) has been proposed to be a key mediator in the hypoxic response of CB chemoreceptors. We hypothesized that H2S contributes to the potentiation of the peripheral chemoreflex response to hypoxia in HF rats. Using the irreversible inhibitor DL‐Propargylglycine (PAG), we tested the effects of inhibition of H2S production on the hypoxic (HVR) and hypercapnic ventilatory responses (HCVR) in HF rats. Experiments were performed on male Sprague‐Dawley rats (200 g) with coronary artery ligation‐induced HF. Ventilatory responses to acute hypoxic (FiO2 = 10%) or hypercapnic (FiCO2 = 7%) stimulation were obtained in unrestrained, freely‐moving rats using whole body plethysmography. HF rats exhibited increased HVR and HCVR compared to control rats. PAG treatment (100 mg/kg IP) reduced the increase in HVR in HF rats (p < .05). However, PAG treatment potentiated the HCVR in HF rats (p < .05). Our results indicate that H2S contributes to the enhanced HVR in HF rats. Moreover, H2S formation also impacts the HCVR suggesting a novel role of H2S on CO2 chemoreception in HF rats.
Depressed serotonin (5‐HT) contributes to suppressed CO2 chemosensitivity in MeCP2 deficient mice
Authors: Toward Marie , Abdala Ana PL , Knopp Sharon J , Paton Julian FR , Bissonnette John M
Published: June 2021

CO2 chemosensitivity is depressed in methyl‐CpG‐binding protein 2 (MeCP2) deficient mice (a pre‐clinical model of Rett syndrome) at low levels of CO2. This deficiency may contribute to their respiratory disorders. Brain levels of 5‐HT are decreased in MeCP2 null males. We hypothesized that increasing central 5‐HT would restore CO2 chemosensitivity. Studies were preformed in B6.129P2(C)‐Mecp2τm1.1Bird null males (MeCP2−/y), wild type (WT) (MeCP2+/y) and heterozygous females (MeCP−/+). Relative to maximum phrenic (PN) amplitude, WT females studied in situ with 3% CO2 in the perfusate had PN of 54.5± 5.8%. In contrast 5 of 8 MeCP2−/+ were apneic at 3% CO2. CO2 chemosensitivity in null males was determined using body plethysmography and exposing the mouse to sequential 1, 3 and 5% CO2. CO2 gain in MeCP2−/y mice was less than that of MeCP2+/y animals (7.18±0.02 vs 9.82±0.1%) (P=<.0.001). Pre‐treatment with citalopram, a selective 5‐HT reuptake inhibitor (2.5 mg/Kg ip) in MeCP2−/y mice resulted in a significant gain in CO2 chemosensitivity to 10.67±0.25% (P= <0.001). These results suggest that decreased serotonin in MeCP2 deficient mice dis‐facilitates CO2 chemosensitivity.

Support: International Rett Syndrome Foundation; Rett Syndrome Research Trust; NewLife Foundation (UK)
The role of Ca2+ and BK channels in the firing rate response of locus coeruleus (LC) neurons to CO2: controlling the chemosensitive gain
Authors: Imber Ann N. , Graham Cathy D. , Putnam Robert W.
Published: June 2021

Chemosensitive LC neurons increase their firing rate in response to CO2 via inhibition of K+ channels. This increase gets smaller during neonatal development (from P3‐P16). Changes of this K+ channel‐based “accelerating” pathway may account for the developmental changes. We used whole cell patch clamp and fluorescence imaging microscopy to study a different basis for these developmental changes in chemosensitive gain, a “braking” pathway. We found that an L‐type Ca2+ current increased in LC neurons during development. This current was activated by CO2, resulting in increases in [Ca2+]i and the activation of hyperpolarizing Ca2+‐activated K+ currents (KCa), which should reduce the firing rate response of LC neurons to CO2. We used the BK (a large conductance KCa) channel inhibitor paxilline and voltage clamp to study this KCa current. Inhibition of the BK current removed the “brake” and increased the firing rate response to CO2 in LC neurons. Notably, the “braking” pathway increased during neonatal development in a fashion that paralleled the decrease in the chemosensitive firing rate response of LC neurons to CO2. These findings suggest a unique role for Ca2+ in controlling central chemoreceptor gain. Abnormalities in this pathway could be associated with disorders involving elevated central gain, such as sleep apnea and panic disorders.

[NIH R01 HL56683, AHA Great Rivers Affiliate Predoctoral Fellowship]
Mitochondrial Content of Carotid Body Type I Cells Decreases During the Maturation of the Hypoxic Response
Authors: Paulet Julia Elizabeth , Tran Huong-Thao , Jordan Heidi , Pye Richard , Ladle David , Wyatt christopher
Published: June 2021

Carotid body (CB) responses to hypoxia are low at birth and increase over time to mature responses. Using an in vitro rat CB‐carotid sinus nerve (CSN) preparation, Kholwadwala and Donnelly (1992) demonstrated that the CSN activity in response to hypoxia increased from low levels to a robust adult response after two weeks. This time course of maturation was paralleled by an increase in TASK channel sensitivity to hypoxia (Kim et al, 2011). The mechanism(s) which underpin this increase in O2‐sensitivity has not been addressed. However, a body of evidence suggest mitochondria in Type I cells are critical for O2‐sensing by the CB. We tested the hypothesis that changes occur in mitochondrial organization during development of O2‐sensitivity in CBs.

Type I cells were isolated from juvenile (4–6 days) and mature rats (14–16 days). Cells were stained with Mitotracker and Celltracker. Total mitochondrial volume in juvenile Type I cells was 100.3 ± 13.6 μm3 (n = 23) and this was significantly smaller in mature cells, 22.6 ± 3.4 μm3 (n = 17, P<0.00003). This decrease in mitochondrial volume was not observed in non‐oxygen sensing cells (superior cervical ganglion cells).

These data demonstrate significant reductions in the mitochondrial content of mature Type I cells. This will likely impact the metabolic setting of the cell and may underpin the development of O2‐sensitivity.

Supported in part by an NIH grant 1RO1HL091836
Hydrogen sulfide mediates catecholamine secretion elicited by hypoxia in the carotid body
Authors: Makarenko Vladislav , Peng Ying-Ji , Nanduri Jayasri , Snyder Solomon H. , Fox Aaron P. , Prabhakar Nanduri R.
Published: June 2021

We previously demonstrated that H2S is a physiologic transmitter in the carotid body, enhancing responses to hypoxia. Carotid bodies express the H2S producing enzyme cystathionine γ‐lyase (CSE). Genetic ablation of CSE or inhibition by DL‐propargylglycine (PAG) eliminated carotid body sensory and ventilatory responses to hypoxia. In the present study we examine the role of H2S in the glomus cell's response to hypoxia. Catecholamine secretion was monitored by carbon fiber amperometry and [Ca2+]i with fura 2. In rat glomus cells PAG attenuated the secretory response to hypoxia, but did not alter secretion elicited by high‐K+. Experiments with CSE−/− mice produced similar results; glomus cells from CSE−/− mice showed little catecholamine secretion in response to hypoxia, but exhibited robust secretion when exposed to high‐K+. Secretion is tightly regulated by [Ca2+]i. PAG treated rat glomus cells exhibited reduced elevation of [Ca2+]i in response to hypoxia. Exposing rat glomus cells to the H2S donor NaHS produced an elevation in [Ca2+]i that mimicked the elevation of [Ca2+]i elicited by hypoxia. More importantly, glomus cells from CSE−/− mice had markedly attenuated elevation of [Ca2+]i in response to hypoxia. These results suggest that hypoxia evoked catecholamine secretion from glomus cells is mediated by H2S via a Ca2+ dependent mechanism. Supported by NIH HL‐90554, HL‐86493.
Clinical Methods to Assess Cardiac Sympathetic Innervation and Function
Authors: Goldstein David S
Published: June 2021

Physiological, pharmacologic, neurochemical, and neuroimaging methods enable evaluation of cardiac sympathetic noradrenergic innervation and function in humans. This lecture provides examples of each approach. Low frequency power of heart rate variability, adjusted for total or high frequency power, has been used to examine cardiac sympathetic “tone” or “sympathovagal balance” but seems related more closely to baroreflex modulation of autonomic outflows than to the outflows themselves. Effects drugs on cardiac contractility can separate pre‐ from post‐ganglionic noradrenergic lesions. Measurement of norepinephrine and its main neuronal metabolite during infusion of tracer‐labeled norepinephrine provides a means to examine key aspects of cardiac sympathetic function in heart failure. Cardiac sympathetic imaging is very powerful for detecting dysfunction or loss of cardiac sympathetic nerves. Using this technology we described cardiac sympathetic denervation in Parkinson disease and obtained evidence for decreased vesicular uptake of intra‐neuronal catecholamines in Lewy body diseases. Clinical laboratory evaluation therefore can provide important clues to the pathophysiology of neurocardiologic disorders.
Calcium Signaling in Induction of Synaptic Plasticity in Cortical Pyramidal Neurons
Authors: Kalikulov Hodja , Friedlander Michael
Published: June 2021

Pairing of pre‐ and postsynaptic activity in layer 2/3 pyramidal neurons in visual cortex at a fixed temporal delay results in variable plasticity outcomes (long term synaptic potentiation ‐ LTP, long term synaptic depression ‐ LTD or no significant change ‐ NC). In order to understand the role that intracellular calcium signaling may play in the different outcomes, we measured [Ca2+]i transients during pairing while pharmacologically inhibiting different calcium sources. In control experiments, pre‐postsynaptic pairing resulted in a net LTP but individual cells in the sample underwent either LTD or LTP or NC. Selective inhibition of individual intracellular calcium sources such as IP3 receptors or ryanodine receptors changed the plasticity outcome to a net LTD when RyRs were blocked with 200μM ryanodine while inhibition of IP3 receptors with 1.0 uM xestospongin resulted in a net LTP. Individual [Ca2+]i. Controls had peak transients =430±87 nM and an average decay time constant = 323±44 msec. However, ryanodine treatment reduced the calcium transient peaks to 310±56 nM and reduced the decay time constant to 239±38 msec. Inhibition of IP3 receptors with xestospongin c reduced the peak transient level to 380±62 nM and the decay time constant to 417±55). Thus, the specific and selective mobilization of different intracellular calcium signaling pathways may be one source of the variable plasticity outcomes between cells.

Supported by NIH grant EY‐12782 to MJF
Gαq/11‐mediated AKT Activation and Intracellular Calcium Responses to Retrograde Flow in Endothelial Cells
Authors: Melchior Benoît , Frangos John A.
Published: June 2021

Temporal gradients in shear stress, including flow reversal, are key actors in the development of dysfunctional endothelial cells (EC) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in [Ca2+]i. We have previously shown that a change in flow direction (retrograde flow) on a flow‐adapted endothelial monolayer induced the realignment of the cell‐cell junction along with a dramatic [Ca2+]i burst. With an enriched expression at the junction Gαq/11 is a strong candidate in shear‐induced increase in [Ca2+]i and was previously shown to be activated within a second after onset of shear stress. In flow‐adapted human EC we have investigated multiple pathways leading to the increase of [Ca2+]i after reversal in flow direction. We observed that the average latency in [Ca2+]i response to a 10 dyn.cm−2 retrograde shear stress was increased by 15 sec in cells silenced for Gαq/11. A similar delay was observed after cells were treated with the PLCβγ inhibitor U73122 or the PI‐PLC inhibitor Edelfosine. Lower levels of IP3 production correlated with the increased latency in [Ca2+]i responses upon the different treatments. Gαq/11 silencing also led to complete abrogation of flow‐induced GSK‐3β and AKT activation. Taken together our results identify Gαq/11 activation as the initial trigger in retrograde flow‐induced [Ca2+]i and a major regulator of the AKT response to shear stress.
Physiological Analysis of the ZO‐1 Actin Binding Region on Cell Adhesion and Function in Epithelial Cells
Authors: Loar Abigail M , Winkler Garry A , Brady Rachel C , King Jonathan M
Published: June 2021

Zonula occludens‐1 (ZO‐1) is a large junctional scaffold protein within epithelium that contributes to the regulation between a proliferative and differentiated state. The function of the ZO‐1 Actin Binding Region (ABR) was examined by overexpression of either GFP‐ABR‐ZO‐1 or 3XFlag‐ABR‐ZO‐1 fusion constructs into stably transfected MDCK II cells. Additionally, 6XHis‐tagged‐ ABR‐ZO‐1 was used to produce recombinant ABR protein to examine molecular interactions with the ABR domain. Calcium switch experiments demonstrated the 3XFlag‐ABR‐ZO‐1 overexpression significantly enhanced barrier function and kinetics of tight junction assembly after an initial lag phase. In live cell imaging experiments, GFP‐ABR‐ZO‐1 markedly altered MDCK cell phenotype into spindle‐like cells deviating from the expected epithelial monolayer and adhesive properties. To study multicellular aggregation in a physiologically relevant system, MDCK cells were induced to form cysts by three‐dimensional cell culture in Geltrex extracellular matrix. GFP‐ABR‐ZO‐1 overexpression showed unusual morphogenesis, with unorganized cysts more than twice the size of control MDCK cysts. These studies highlight the ZO‐1‐ABR region as an important regulator of cell morphology, adhesion, and multicellular epithelial organization.
Functional significance of CD44 and MMP‐9 in cartilage homeostasis
Authors: Askew Emily B , Warren Kelly M , Knudson Cheryl B , Knudson Warren
Published: June 2021

Previous reports have proposed a mechanistic link between the CD44 cell surface receptor and the matrix metalloproteinase (MMP)‐9 in tumor invasion, prostate cancer progression and osteoclast migration. CD44 is expressed by articular chondrocytes and interestingly during the progression of osteoarthritis (OA) CD44 fragmentation occurs, resulting in a reduction in hyaluronan (HA) binding capacity. Furthermore, both treatment with the inflammatory cytokine and catabolic agent IL‐1β, as well as disruption of CD44‐HA interactions result in stimulation of MMP‐9. Thus, the purpose of this study is to determine CD44‐dependent, MMP‐9 related signaling events with the goal of identifying novel OA therapeutic targets. Primary chondrocytes and femoral heads were isolated from 7–14 day old BALB/c wild‐type (WT) and CD44 knock‐out (KO) mice. Treatment with IL‐1β showed a loss in safranin O staining of the extracellular matrix only in treated femoral heads from WT mice. Immunoblot analysis and zymography revealed that MMP‐9 expression and gelatinase activity is higher in chondrocytes obtained from WT mice that were treated with IL‐1β versus CD44 KO IL‐1β‐treated cells. Taken together, these studies suggest a functional link between CD44 expression status, cell associated HA and MMP‐9 activity in cartilage. Supported by NIH R01‐AR39507 and R01‐AR43384.
A Morpho‐Etiological Work‐Up of Congenital Limb Anomalies
Authors: Tayel Shawky Mahmoud , Ismail Hanaa , Abd Rabuh Abdel Rahman , Tayel Sally , Sallam Hassan , Naguib Kamal
Published: June 2021

BACKGROUND

Limb anomalies rank behind congenital heart disease as the most common birth defects observed in infants. More than 50 classifications for limb anomalies based on morphology and osseous anatomy have been drafted over the past 150 years. The present work aims to provide a concise summary of the most common congenital limb anomalies on a morpho‐etiological basis.

PATIENTS AND METHODS

In a retrospective study, 81 newborns with anomalies of the upper and/or lower limbs were ascertained through clinical examination, chromosomal analysis, skeletal surveys and other relevant investigations.

RESULTS

Fetal causes of limb anomalies represented 75.3% of the cases in the form of 15 cases (18.5%) with chromosomal aberrations (trisomy 13, 18 and 21, duplication 13q and deletion 22q) and 33 cases (40.7%) with single gene disorders. An environmental etiology for limb anomalies was diagnosed in 13 cases (16.1%) as amniotic band disruption, monozygotic twin with abnormal circulation, vascular disruption (Poland sequence, sirenomelia and general vascular disruption) and an infant with a diabetic mother, Twenty cases (24.7%) had limb anomalies as part of sporadic syndromes of unknown etiology.

CONCLUSIONS

The morpho‐etiological work‐up of limb anomalies adopted in the present study is valuable for detecting the cause of the anomaly and is crucial for its prevention. Prevention can be achieved by proper genetic counseling, which includes recurrence risk estimation and prenatal diagnosis.
The use of mesenchymal stromal cells improves wound repair in rat abdominal incisions
Authors: Minteer Tanya Elise , Heffner Jeremy , Holmes Jonathan , Ferrari Jonathan , Krontiris-Litowitz Johanna , Marie Hazel , Perko John , Dorion Heath , Fagan Diana
Published: June 2021

More than 200,000 abdominal hernias are repaired each year. Although new surgical techniques and technologies have attempted to combat the occurrence of hernias, patients continue to suffer from hernias following abdominal surgeries. Regenerative therapy, including the use of bone marrow derived‐mesenchymal stromal cells (MSCs), offers a new avenue to reducing the problem of hernias seen in post‐operative patients. Midline laparotomies were performed on three groups of Lewis rats. Group 1 rats were designated as the control group and were repaired without additives. CollaTape containing platelet‐rich plasma (PRP) was used to repair the abdominal incisions of group 2 rats. The incisions of group 3 rats were repaired similarly to group 2 but with the addition of MSCs. At 4 and 8 weeks postoperative, abdominal fascia was excised (n=7 per group). Collagen deposition and organization (as determined by a Trichrome stain) was rated by blinded observers. Group 3 showed significantly increased neovascularization and collagen deposition compared to the abdominal fascia of groups 1 and 2. The results of this study suggest that the use of MSCs in abdominal repairs could serve as a beneficial tool to improve wound healing.

Grant Funding Source : LifeCell Hernia Resident/Fellow Research Grant
Design of bioactive smart nanofilms with bFGF and PDGF‐BB for wound repair
Authors: Challagundla Sukeerth , Mills David K.
Published: June 2021

Wound healing has become a prominent area of research in regenerative medicine. Although many wound healing and wound dressing techniques have been developed, there remains a great demand for an enhanced treatment modality. Fibroblast Growth Factor (bFGF) and Platelet Derived Growth Factor –BB (PDGF– BB) were incorporated into a polyelectrolyte multilayer nanocoating (PMNs) and applied to a wounded skin equivalent (Epiderm™). The nanocoating consisted of a ten‐layer Poly‐L‐Lysine (PLL) and Poly‐Glutamic‐Acid (PGA) nanocoating with growth factors embedded in two different modalities, a deep layer and double layer. Tissues treated with the designed PMNs enhanced re‐epithelialization, collagen synthesis and the rate of wound healing significantly. The degree of tissue response varied with each nanocoating. The double layer modality showed improved healing compared to the single modalities with an increase in cell number at the wound site with greater proliferation in the epidermis compared to the dermis. Picrosirius Red staining results showed an increase in the level of collagen deposition in the dermis. Controls, with a ten‐layer nanocoating, and no growth factors, showed marginal wound repair. These results suggest that bioactive nanocoatings may have potential as a treatment modality for cutaneous and chronic wounds.

Funding provided by the State of Louisiana Governor's Biotechnology Initiative.
Bioactive Hydrogels for TMJ Repair
Authors: Karnik Sonali Jayant , Mills David
Published: June 2021

The temporomandibular joint (TMJ) is a biaxial joint that functions in articulation, food acquisition and mastication. According to the National Institutes of Health, 3–5% population of the American population suffers from some form of TMJ disorder (TMJD). This study's objective was to develop a bioactive hydrogel that is osteoconductive, osteogenic, biodegradable and will mimic the internal environment of the TMJ. Calcium alginate hydrogels (2%) and halloysite nanotubes (HNTs) (1%) nanocomposites were loaded with Bone Morphogenetic Protein 2 (BMP2). Osteoblasts were encapsulated within these hydrogel nanocomposites. These constructs were cultured over a 21‐day period and cell behavior assessed for cell proliferation, functionality, mineralization, etc. HNTs released BMP2 in a sustained fashion and enhanced osteocyte differentiation and tissue formation as compared with controls (+BMP2/no HNT or HNTs only). Cell proliferation, mineralization and protein content increased over the experimental period. Our results suggest that HNT/alginate hydrogel composites may serve as a viable repair system for TMJDs. Funding provided by the State of Louisiana Governor's Biotechnology Initiative.
Tissue engineering bone by recapitulating developmental and repair programs offers improved biological outcomes
Authors: Bahney Chelsea Shields , Hu Diane , Miclau Theodore , Marcucio Ralph
Published: June 2021

The principle behind tissue engineering and regenerative medicine is development of functional replacements for damaged or diseased tissues. In the case of bone, technologies focus on promoting direct osteogenesis. However, current therapies are associated with clinical drawbacks, including osteonecrosis and poor integration. Bone regeneration through endochondral ossification, which is the mechanism by which long bones form and the majority of fractures heal, may overcome these problems. We hypothesize that cartilage grafts can promote a vascularized and integrated bone regenerate. To test this, we created stabilized defects in the mid‐diaphysis of the murine tibia and transplanted cartilage grafts from reporter mice. The cartilage was replaced by vascularized bone that integrated with the host. Since endochondral ossification is thought to require chondrocyte apoptosis and cartilage replacement by osteoblasts from the invading vasculature, we predicted the new bone would be host‐derived. However, the new bone was composed largely of donor‐derived cells and there was limited evidence of apoptosis. Together, these data suggest that in our animal model an alternative healing mechanism, such as transdifferentiation, is occurring. Translation of these data into a patient‐based therapy provides a platform to improve clinical outcomes and gain insight into the biology of bone regeneration.

Grant Funding Source: NIH‐NIAMS (R01‐AR053645‐01 to TM), UCSF GEMS‐CSTI Fellowship (CSB)
Denritic Morphology
Authors: WHITE EDWARD L
Published: June 2021

The common assumption that dendrites are cylindrical is incorrect. Analyses of spiny stellate cell dendrites reconstructed from serial thin sections through mouse barrels shows these dendrites to be flattened; the ratio of long to short axes in cross‐section is typically 2:1. Planes of flattening are unrelated to the orientation of dendritic segments, are not related to plane of section, and are unchanged for various estimates of section thickness. Thus, the flattened shape is due neither to tissue compression, sectioning artifact, nor to inaccuracy of reconstruction. Two‐thirds of dendritic spines are attached at the narrow, ridge‐like regions of the flattened shafts. Apical dendrites of pyramidal cells are also flattened and exhibit a preferential distribution of spines. These phenomena may be related to the distribution of cytoskeletal elements. The flattened shape may be an artifact of fixation, and if so, could be imagined to result from the shrinking of cell membrane around the cytoskeleton and/or from the collapse of cytoskeletal elements within certain compartments. Alternatively, the shape may reflect the in vivo condition. Consequences for various aspects of electrical activity within dendrites of the possible compartmentalization of the dendroplasm and of the observed irregular distribution of spines remain to be determined. Israel Science Foundation grant 52/00‐4
Auditory‐amygdalar circuits: An anterograde and retrograde tracing study
Authors: Peterson Diana C
Published: June 2021

The amygdala, a brain region that determines stimulus significance, receives auditory information from medial geniculate and auditory cortex and projects back to the auditory system through direct and indirect circuits. The current study utilized retrograde tracing to identify the location of amygdalo‐auditory cortex neurons within the amygdala. Labeled neurons were primarily pyramidal and were located throughout the lateral and basal nuclei of the amygdala. Previous studies have determined that the pyramidal projection neurons are glutamatergic. Therefore a large percentage of this population is likely to have excitatory influences on the target neurons. To determine the termination pattern of these neurons, anterograde tracing studies were performed. We observed both en passant and terminal boutons throughout layers I through IV of primary and secondary auditory cortex. Boutons were much more numerous in layers V and VI, and seemed to target both pyramidal and non‐pyramidal neurons. Although the influence of these projections is yet unknown, the widespread terminal distribution we observed indicates that amygdalar projections influence numerous cortical and sub‐cortical auditory circuits. We hypothesize that these projections alter auditory processing at numerous levels of the auditory system toward relevant environmental cues. Supported by NIDCD R03 DC010245.
In vitro generated microglia derived from blood precursors
Authors: Etemad Samar , Ruitenberg Marc , Filgueira Luis
Published: June 2021

Microglia are the macrophages of the central nerve system expressing various markers of the monocyte‐macrophage lineage such as surface molecules, chemokine/cytokine receptors, and displaying corresponding functions, including phagocytosis and antigen presentation. The aim of this study is to establish a new in vitro microglia model using blood‐derived precursor cells.

For that purpose, human blood derived monocytes were cultured in the presence of a panel of cytokines and chemokines to generate in vitro microglia. The cells were further characterized for morphological, functional and phenotypic properties and compared with monocytes and in vitro generated dendritic cells. Monocytic lineage marker (CD45, CD14, HLA‐ DR) and chemokine receptors CCR1, CCR2, CCR4, CCR5, CXCR1 and CXCR3 were studied. In vitro generated microglia cells were different from monocytes and dendritic cells in morphology, function and expression of surface markers. In comparison with immature dendritic cells, microglia showed lower capacity in phagocytosing bacteria, antigen presentation and T lymphocyte stimulation.

In conclusion, a new protocol for in vitro culture of human microglia cells has been established. Further studies to characterize the microglia at molecular level and use them for functional and toxicity studies are ongoing.

Grant Funding Source: School funding
Lectin‐mediated defense of the intestinal epithelial surface
Authors: Hooper Lora , Vaishnava Shipra , Mukherjee Sohini
Published: June 2021

The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host‐bacterial relationships are maintained without eliciting potentially harmful immune responses. We discovered that resident intestinal bacteria trigger epithelial cell expression of RegIIIgamma, a member of the C‐type lectin family of carbohydrate binding proteins. RegIIIgamma directly kills Gram‐positive bacteria and thus represents a novel class of antibacterial proteins. We have elucidated the mechanistic basis for RegIIIgamma bactericidal activity by showing that it kills bacteria by forming an oligomeric pore in the bacterial inner membrane. Using cryoelectron microscopy, we determined the structure of the hexameric pore assembly at subnanometer resolution. In vivo studies have revealed that RegIIIgamma is essential for maintaining a ~50 micron zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host‐bacterial segregation in RegIIIgamma−/− mice is coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Together, our findings reveal that RegIIIgamma is a fundamental immune mechanism that promotes host‐bacterial mutualism by regulating the spatial relationships between microbiota and host.
Mechanism of TRAF6 Intranuclear Translocation
Authors: Su An-Jey , Adamik Juraj , Sydeski-Hurd Jennifer , Auron Philip E.
Published: June 2021

Proper cellular response to external signals requires exact spatiotemporal positioning of intracellular components. Adaptor molecules are used to assemble protein complexes to facilitate signaling pathways. We studied the interplay between two adaptor molecules, tumor necrosis factor receptor associated factor‐6 (TRAF6) and Syntenin (Syn‐1). TRAF6 transduces signaling for the TNF and the Toll‐like/Interleukin receptor families. Many studies focused on cytoplasmic TRAF6, but few report on the inhibition of TRAF6 and the mechanisms by which it enters the nucleus. We find that overexpression of Syn‐1 not only attenuates TRAF6 signaling, but causes nuclear localization in both HEK293 and RAW246.7 cells. TRAF6 is an E3‐ligase whose activity we previously reported is maintained by auto‐ubiquitination and an open conformation. Knockdown of Syn‐1 potentiates TRAF6 activation and ubiquitination. Syn‐1 exhibits reduced effect on TRAF6 mutants that cannot be ubiquitinated. This implies that Syn‐1 binds to TRAF6 in a closed state. Protein domain deletions in both TRAF6 and Syn‐1 show that the full‐length molecules are not necessary for interaction. Mutagenesis or deletion of a newly identified TRAF Interaction motif (TIM) in Syn‐1 reveals differences in how Syn‐1 affects TRAF6 signaling and localization. Syn‐1 also localizes to the nucleus when overexpressed with either wild type or a constitutively active TRAF6. Lastly, Syn‐1 binds to the transcription factor Sox4 resulting in Sox4‐Syn‐1 relocalization to the nucleus. We have observed that overexpression of Sox4 also induces TRAF6 nuclear translocation, providing a mechanism for TRAF6 entry into the nucleus.
Use of qChIP to identify genes targeted by the Ikaros tumor suppressor
Authors: Payne Jonathon L , Song Chunhua , Ortiz Nathalie Fuentes , Sloane Justin , Payne Kimberly J , Payne Marvin A , Dovat Sinisa
Published: June 2021

The IKAROS gene encodes a DNA binding zinc finger protein that is essential for hematopoietic differentiation and acts as a tumor suppressor in leukemia. The Ikaros protein is known to both activate and repress target gene expression and to participate in chromatin remodeling. The specific mechanisms by which Ikaros exerts it tumor suppressor activities are unknown. We are using Chromatin Immunoprecipitation (ChIP) assay combined with quantitative PCR (qPCR), called qChIP, to identify genes whose expression is directly regulated by Ikaros. Using this technique we calculate the difference in Ikaros binding to the promoter region of the target genes versus nonspecific binding (control). qChIP assays show that Ikaros binds to the promoter regions of the MYC oncogene, a known Ikaros target, and two other novel target genes. The promoter regions of MYC and the two novel target genes were cloned into luciferase reporter constructs and used to transiently transfect HEK cells that were co‐transfected with Ikaros or empty vector. Luciferase reporter assays showed that Ikaros suppresses the promoter activity of MYC and the two novel target genes. Our data confirm that the MYC oncogene is an Ikaros target and identify two novel Ikaros target genes. These data suggest that Ikaros exerts its tumor suppression function by inhibition of target gene expression in leukemia cells.

Supported by R01 HL095120, a St. Baldrick's Foundation Career Development Award, the Four Diamonds Fund of Pennsylvania State University, College of Medicine (to SD), the NIDDK Short‐term Education Program for Under‐Represented Persons (to NFO) and a La Sierra University Dept. of Chemistry and Biochemistry stipend (to JLP).
TRF2 regulates differential DNA damage response signaling from intermediate‐state and uncapped‐state telomeres
Authors: Cesare Anthony J , Hayashi Makoto T , Karlseder Jan
Published: June 2021

There are three states of human telomere protection: a fully protected “closed‐state”, a DNA damage response (DDR) positive but fusion resistant “intermediate‐state” and a fusion competent “uncapped‐state”. We developed a system to induce intermediate‐or uncapped‐state telomeres in human primary and cancer cells through a graduated depletion of TRF2. We found intermediate‐state telomeres induce a localized ATM‐dependent DDR. However, TRF2 retained at the DDR‐positive chromosome end prevents RNF8 and RNF168 dependent fusions by inhibiting repair at a step between 53BP1 loading onto chromatin and telomeric 3′ overhang removal. Despite being γ‐H2AX positive, intermediate‐state telomeres fail to activate CHK2 and pass through G2 and M to induce p53‐dependent G1 arrest. Fusogenic uncapped‐state telomeres, on the contrary, trigger ATM‐CHK2 signaling analogous to double‐strand breaks. We conclude that TRF2 regulates a specialized chromatin environment by restricting DDR signaling and repair to maintain genomic stability in aged primary cells with spontaneous intermediate‐state telomeres. The NIH, the Human Frontier Science Program and the Japan Society for the Promotion of Science supported this work.
The Disruption of the Mitotic Cell Cycle by Inositolless Death in Saccharomyces cerevisiae
Authors: Hanson Barbara Ann , O'Connor Paul , Munezero Jean , Croglio Vincent
Published: June 2021

When an inositol‐requiring mutant (MC6A) of Saccharomyces cerevisiae undergoes inositolless death, there is a rapid loss of cell viability. There also is a significant decline in the transcription of CLB2 mRNA levels. CLB2p activates a cyclin‐dependent kinase (CDK) to promote the progression from G2 into the M phase. CDK activity eventually decreases through CLB2 degradation which is caused by the Anaphase‐Promoting Complex (APC). In these cells, the spindle apparatus may form but may not receive the signals to depolymerize at the end of the cycle. To test this idea, changes in polymerized tubulin in the spindle apparatus in these cells were examined by staining the cells with TubulinTracker™ (Oregon Green® 488 Taxol bis‐acetate). Changes in TUB1, CDC28, CDC20, and APC1‐4 mRNA transcription also were measured by RT‐PCR 80 % of the budding cells stained in cells after 24h of inositolless death; however, there was a major increase in the proportion of stained nonbudding cells (54.4%). This was a 2.5 fold increase compared to control cells. The levels of transcribed TUB1 mRNAs also did not decline during inositolless death. The tubulin subunits may be polymerizing to form the spindle apparatus during inositolless death in these cells, but the results suggest that the cellular signals to depolymerize the tubulin may be disrupted because of the reduced levels of CLB2p which affect its degradation by APC.
Blocking ubiquitin deposition at telomeres: the molecular mechanism of TRF2‐mediated end protection
Authors: Okamoto Keiji , Denchi Eros Lazzerini
Published: June 2021

“End protection” defines that ability of telomeres to cap and protect chromosome ends from the DNA damage response machinery. When end protection is lost, chromosome ends are recognized as sites of DNA lesion and are repaired by the DNA repair machinery resulting in end‐to‐end chromosome fusions, an unstable genomic configuration. The telomere repeat binding factor TRF2 plays a crucial role in this process as shown by the fact that its depletion is sufficient to trigger activation of DNA damage response at chromosome ends. In order to uncover the molecular mechanism employed by TRF2 to protect chromosome ends we performed a screen to identify the critical region within TRF2 involved in end protection. Our data show that a critical mechanism for TRF2‐mediated suppression of DNA damage activation involves suppression of RNF168‐dependent ubiquitination of histone H2A(X). Prevention of this histone modification is both required and sufficient to suppress activation of DNA damage response at chromosome ends. Furthermore, our data reveal a critical role for de‐ubiquitinase enzymes for telomere integrity.

ADI
Telomerase activity, HPV presence and splice pattern of hTERT mRNA in cervical intraepithelial neoplasias and adjacent normal tissues
Authors: Skvortsov Dmitry Alexandrovich , Petrenko Anatolii Anatolevich , Korolenkova Lubov Ivanovna , Zvereva Maria Imilievna , Rubtsova Maria Petrovna , Kisseljov Fedor Ljvovich , Dontsova Olga Anatoljevna
Published: June 2021

Cervical cancers are characterized by the persistence of human papilloma virus (HPV) genome that is found in tissue samples starting from the early stages of tumor progression. Just like in other tumors, the activation of telomerase was observed in cervical carcinomas, but information about its expression was controversial. On model systems (cell lines) alternate splicing of human telomerase reverse transcriptase mRNA may take part in telomerase activity regulation.

The aim of this study was to find possible correlations between the presence of HPV sequences, activity of telomerase and expression of different spliced forms of hTERT RNA in cervical intraepithelial neoplasias (CIN). The results show that HPV DNA is present in 60% of normal tissue adjacent to CIN lesions and up to 84% in CIN samples. Telomerase activity was found in 28% of adjacent normal tissue and in 68% of CIN II–III. hTERT RNA that encodes an active enzyme was present almost in all CIN samples. Variations in levels of telomerase activity are presumably not regulated by the splicing forms of hTERT mRNA with deletions.
Integrating Undergraduate Research/Research Training into the Curriculum at Research‐Intensive Institutions
Authors: Kennelly Peter James
Published: June 2021

Educators, employers, and admissions committees all place a high value upon undergraduate research experiences beyond the required laboratory courses. Undergraduate research experiences immerse the students in the environment of a working research, production, or analytical laboratory that amplifies the importance of independence, initiative, and communication skills. At many schools the ratio of faculty to students is such that each and every student can be required to do some sort of independent research project. However, at large, research‐intensive the ratio of faculty to students in the Biochemistry Department frequently ranges from 10 to 40:1. Finding placements within the departmental faculty for all interested students can be impractical. In this presentation, we will talk about the many potential sources of research mentors that lie outside traditional department boundaries at an R1 university, potential pitfalls, and ways of addressing them.