Comparison of dosimetric data of bone marrow between standard IMRT and bone marrow sparing IMRT in carcinoma cervix

BackgroundThe aim of the study was to assess the dosimetric comparison of bone marrow between standard IMRT(SD-IMRT) and bone marrow sparing IMRT (BMS-IMRT) among carcinoma cervix patients who underwent radical or adjuvant chemoradiation in a tertiary cancer center.Materials and methodsForty eligible patients of histo-pathologically proven carcinoma cervix were enrolled in the study that was randomized on a 1:1 basis between SD-IMRT and BMS-IMRT from July 2018 to October 2019. The whole pelvis, bilateral femoral heads, and upper 1/3^rd femur were contoured using the whole bone technique as a surrogate marker for the bone marrow. In both arms, V10, V20, and V40, bone marrow was noted along with mean, maximum, minimum dose, and total volume. DVH for the bone marrow in both arms was compared using the unpaired student t-test.ResultsWe found no significant difference in the mean of various parameters in SD-IMRT arm vs. BMS IMRT arm — for the bone marrow: V10 (89 ± 4.3% vs. 86.7 ± 3.7%), V20 (73.2 ± 5.3% vs. 73.1 ± 4.5%), V40 (23.9 ± 5.4% vs. 26.6 ± 7.4%) and, similarly, for mean dose (28.1 ± 3.5% vs. 28.1 ± 1.8%), maximum dose (53.4 ± 0.58% vs. 53.2 ± 0.58%), minimum dose (0.33 ± 0.18% vs. 0.38 ± 0.38%), total volume (961 ± 110 cc vs. 901 ± 152 cc).ConclusionThis study shows no statistically significant difference in dosimetry between the two groups, which suggests that SD-IMRT spares the bone marrow adequately. Therefore, the need for BMS-IMRT using the present contouring technique does not give any added advantage over SD-IMRT. However, large sample size, other novel contouring technique, and multivariate analysis are needed to reach a definite conclusion.     

Antenatal Dexamethasone after Asphyxia Increases Neural Injury in Preterm Fetal Sheep

Background and Purpose

Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain.

Methods

Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach.

Results

Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290±76 vs 484±98 neurons/mm², mean±SEM, P<0.05) and basal ganglia (putamen, 538±112 vs 814±34 neurons/mm², P<0.05) compared to asphyxia-saline, and with greater loss of both total (913±77 vs 1201±75/mm², P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66±8 vs 114±12/mm², P<0.05, vs sham controls 165±10/mm², P<0.001). This was associated with transient hyperglycemia (peak 3.5±0.2 vs. 1.4±0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum −1.5±1.2 dB vs. −5.0±1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures.

Conclusions

In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.     

Anterograde Degeneration along the Visual Pathway after Optic Nerve Injury

Purpose

To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.

Methods

Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.

Results

An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm², P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10² cells/mm², P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10² cells/mm², P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm², P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10² cells/mm², P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.

Conclusions

Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.     

Increased Short-Term Beat-To-Beat Variability of QT Interval in Patients with Acromegaly

Cardiovascular diseases, including ventricular arrhythmias are responsible for increased mortality in patients with acromegaly. Acromegaly may cause repolarization abnormalities such as QT prolongation and impairment of repolarization reserve enhancing liability to arrhythmia. The aim of this study was to determine the short-term beat-to-beat QT variability in patients with acromegaly. Thirty acromegalic patients (23 women and 7 men, mean age±SD: 55.7±10.4 years) were compared with age- and sex-matched volunteers (mean age 51.3±7.6 years). Cardiac repolarization parameters including frequency corrected QT interval, PQ and QRS intervals, duration of terminal part of T waves (T_peak-T_end) and short-term variability of QT interval were evaluated. All acromegalic patients and controls underwent transthoracic echocardiographic examination. Autonomic function was assessed by means of five standard cardiovascular reflex tests. Comparison of the two groups revealed no significant differences in the conventional ECG parameters of repolarization (QT: 401.1±30.6 ms vs 389.3±16.5 ms, corrected QT interval: 430.1±18.6 ms vs 425.6±17.3 ms, QT dispersion: 38.2±13.2 ms vs 36.6±10.2 ms; acromegaly vs control, respectively). However, short-term beat-to-beat QT variability was significantly increased in acromegalic patients (4.23±1.03 ms vs 3.02±0.80, P<0.0001). There were significant differences between the two groups in the echocardiographic dimensions (left ventricular end diastolic diameter: 52.6±5.4 mm vs 48.0±3.9 mm, left ventricular end systolic diameter: 32.3±5.2 mm vs 29.1±4.4 mm, interventricular septum: 11.1±2.2 mm vs 8.8±0.7 mm, posterior wall of left ventricle: 10.8±1.4 mm vs 8.9±0.7 mm, P<0.05, respectively). Short-term beat-to-beat QT variability was elevated in patients with acromegaly in spite of unchanged conventional parameters of ventricular repolarization. This enhanced temporal QT variability may be an early indicator of increased liability to arrhythmia.     

Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiomyocyte Hypertrophy

Enhanced expression and activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.     

“Immunonutrition” Has Failed to Improve Peritonitis-Induced Septic Shock in Rodents

Background

Immunonutrition in sepsis, including n-3 poly-unsaturated fatty acids (PUFAs) or L-arginine supplementation, is a controversial issue that has yielded a great number of studies for the last thirty-five years, and the conclusions regarding the quantity and quality of this support in patients are deceiving. The aim of the present experimental study is to investigate the effects of a pretreatment with enteral nutrition enriched with n-3 PUFAs or L-arginine on vascular dysfunctions, inflammation and oxidative stress during septic shock in rats.

Design

Rats were fed with enteral Peptamen^® HN (HN group), Peptamen^® AF containing n-3 PUFAs (AF group) or Peptamen^® AF enriched with L-arginine (AFA group). On day 4, peritonitis by cecal ligation and puncture (CLP) was performed. Rats were resuscitated (H18) once septic shock was established. After a 4-hour resuscitation, vessels and organs were harvested to assess inflammation, superoxide anion, nitric oxide and prostacyclin levels. Ex-vivo vascular reactivity was also performed.

Results

Compared to CLP-AF or CLP-HN groups, 47.6% of CLP-AFA rats died before the beginning of hemodynamic measurements (vs. 8.0% and 20.0% respectively, p<0.05). AF and AFA rats required significantly increased norepinephrine infusion rates to reach the mean arterial pressure objective, compared to CLP-HN rats. Both CLP-AF and CLP-AFA reduced mesenteric resistance arterial contractility, decreased vascular oxidative stress, but increased NF-κB (0.40±0.15 in CLP-AF and 0.69±0.06 in CLP-AFA vs. 0.09±0.03 in SHAM rats and 0.30±0.06 in CLP-HN, ß-actin ratio, p<0.05) and pIκB expression (0.60±0.03 in CLP-AF and 0.94±0.15 in CLP-AFA vs. 0.04±0.01 in SHAM rats and 0.56±0.07 in CLP-HN, ß-actin ratio, p<0.05), nitric oxide and prostacyclin production in septic rats.

Conclusions

Although n-3 PUFAs or L-arginine supplementation exhibited an antioxidant effect, it worsened the septic shock-induced vascular dysfunction. Furthermore, mortality was higher after L-arginine supplementation.     

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca²⁺ATPases (SERCAs) are the main Ca²⁺ pumps which decrease the intracellular Ca²⁺ level by reaccumulating Ca²⁺ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca²⁺ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca²⁺-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca²⁺]i measured at the 20^th and 40^th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca²⁺-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca²⁺]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.     

Chronic Lead Exposure Increases Blood Pressure and Myocardial Contractility in Rats

We investigated the cardiovascular effects of lead exposure, emphasising its direct action on myocardial contractility. Male Wistar rats were sorted randomly into two groups: control (Ct) and treatment with 100 ppm of lead (Pb) in the drinking water. Blood pressure (BP) was measured weekly. At the end of the treatment period, the animals were anaesthetised and haemodynamic parameters and contractility of the left ventricular papillary muscles were recorded. Blood and tissue samples were properly stored for further biochemical investigations. Statistical analyses were considered to be significant at p<0.05. The lead concentrations in the blood reached approximately 13 µg/dL, while the bone was the site of the highest deposition of this metal. BP in the Pb-treated group was higher from the first week of lead exposure and remained at the same level over the next four weeks. Haemodynamic evaluations revealed increases in systolic (Ct: 96±3.79 vs. Pb: 116±1.37 mmHg) and diastolic blood pressure (Ct: 60±2.93 vs. Pb: 70±3.38 mmHg), left ventricular systolic pressure (Ct: 104±5.85 vs. Pb: 120±2.51 mmHg) and heart rate (Ct: 307±10 vs. Pb: 348±16 bpm). Lead treatment did not alter the force and time derivatives of the force of left ventricular papillary muscles that were contracting isometrically. However, our results are suggestive of changes in the kinetics of calcium (Ca⁺⁺) in cardiomyocytes increased transarcolemmal Ca⁺⁺ influx, low Ca⁺⁺ uptake by the sarcoplasmic reticulum and high extrusion by the sarcolemma. Altogether, these results show that despite the increased Ca⁺⁺ influx that was induced by lead exposure, the myocytes had regulatory mechanisms that prevented increases in force, as evidenced in vivo by the increased systolic ventricular pressure.     

Advanced Glycation End Products Impair Voltage-Gated K+ Channels-Mediated Coronary Vasodilation in Diabetic Rats

Background

We have previously reported that high glucose impairs coronary vasodilation by reducing voltage-gated K⁺ (K_v) channel activity. However, the underlying mechanisms remain unknown. Advanced glycation end products (AGEs) are potent factors that contribute to the development of diabetic vasculopathy. The aim of this study was to investigate the role of AGEs in high glucose-induced impairment of K_v channels-mediated coronary vasodilation.

Methods

Patch-clamp recording and molecular biological techniques were used to assess the function and expression of K_v channels. Vasodilation of isolated rat small coronary arteries was measured using a pressurized myograph. Treatment of isolated coronary vascular smooth muscle cells (VSMCs) and streptozotocin-induced diabetic rats with aminoguanidine, the chemical inhibitor of AGEs formation, was performed to determine the contribution of AGEs.

Results

Incubation of VSMCs with high glucose reduced K_v current density by 60.4 ± 4.8%, and decreased expression of K_v1.2 and K_v1.5 both at the gene and protein level, whereas inhibiting AGEs formation or blocking AGEs interacting with their receptors prevented high glucose-induced impairment of K_v channels. In addition, diabetic rats manifested reduced K_v channels-mediated coronary dilation (9.3 ± 1.4% vs. 36.9 ± 1.4%, P < 0.05), which was partly corrected by the treatment with aminoguanidine (24.4 ± 2.2% vs. 9.3 ± 1.4%, P < 0.05).

Conclusions

Excessive formation of AGEs impairs K_v channels in VSMCs, then leading to attenuation of K_v channels-mediated coronary vasodilation.     

Development and Characterization of Mixed Niosomes for Oral Delivery Using Candesartan Cilexetil as a Model Poorly Water-Soluble Drug

The aim of this study was to prepare candesartan cilexetil-loaded niosomes and mixed niosomes to enhance the aqueous solubility of the drug, thus improving its oral bioavailability. The formulations were prepared using various types and combinations of surfactants, copolymers, and charge-inducing agents. The candesartan cilexetil entrapment efficiency, particle size, and zeta potential of these niosomes varied within the range of 99.06 ± 1.74 to 36.26 ± 2.78, 157.3 ± 3.3 to 658.3 ± 12.7 nm, and −14.7 ± 2.8 to −44.5 ± 1.5 mV, respectively. The in vitro drug release from niosomes was improved after niosomal entrapment compared to pure candesartan cilexetil. The sedimentation behavior study and formulation stability tests against bile salt revealed that mixed niosomes prepared by combining Span 60 and Pluronic P85 demonstrated better stability. The differential scanning calorimetry analysis showed the conversion of crystal structure of candesartan cilexetil to the soluble amorphous form after niosomal encapsulation which induced the drug release. Consequently, oral drug delivery by Span 60/Pluronic P85-mixed niosomes seems feasible due to enhanced drug release and stability.KEY WORDS: in vitro drug release, niosomes, oral drug delivery, stability, surfactants     

Sprint Interval and Sprint Continuous Training Increases Circulating CD34+ Cells and Cardio-Respiratory Fitness in Young Healthy Women

Introduction

The improvement of vascular health in the exercising limb can be attained by sprint interval training (SIT). However, the effects on systemic vascular function and on circulating angiogenic cells (CACs) which may contribute to endothelial repair have not been investigated. Additionally, a comparison between SIT and sprint continuous training (SCT) which is less time committing has not been made.

Methods

12 women (22±2 yrs) completed 12 sessions of either SIT (n = 6) or work-matched SCT (n = 6) on 3 days/week. Pre and post-training assessments included brachial artery endothelial function and peripheral blood analysis for CAC number (CD34⁺/CD34⁺CD45^dim). CAC function was measured by migration and adhesion assays. Cardio-respiratory fitness, carotid arterial stiffness and carotid-radial and brachial-foot pulse wave velocity (PWV) were also evaluated.

Results

CD34⁺ CACs increased following training in both groups but CD34⁺CD45^dim did not (Pre CD34⁺: 40±21/10⁵ leukocytes, Post CD34⁺: 56±24/10⁵ leukocytes, main time effect p<0.05). Brachial artery flow-mediated dilation (FMD) increased following SIT but SCT had no effect (Pre SIT: 5.0±3.4%, Post SIT: 5.9±3.0%, Pre SCT: 7.2±2.7%, Post SCT: 6.5±2.9%; group x time interaction p = 0.08). increased in both training groups (Pre: 34.6±4.6 ml•kg•ml⁻¹, Post: 36.9±5.4 ml•kg•ml⁻¹, main time effect p<0.05). CAC function, carotid arterial stiffness and PWV did not change after training (p>0.05).

Discussion

SCT involving little time commitment is comparable to SIT in increasing CD34⁺ cell number and . An increased mobilisation of CD34⁺ CACs suggests that sprint training may be an effective method to enhance vascular repair.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via a nongenomic PPARγ-dependent pathway

Oxidized phospholipids have been shown to exhibit pleiotropic effects in numerous biological contexts. For example, 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxidized phospholipid formed from alkyl phosphatidylcholines, is a peroxisome proliferator–activated receptor gamma (PPARγ) nuclear receptor agonist. Although it has been reported that PPARγ agonists including thiazolidinediones can induce plasma volume expansion by enhancing renal sodium and water retention, the role of azPC in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues and also investigated the effect of azPC on renal sodium handling in vivo. We showed using a microperfusion technique that azPC rapidly stimulated Na⁺/HCO₃⁻ cotransporter 1 (NBCe1) and luminal Na⁺/H⁺ exchanger (NHE) activities in a dose-dependent manner at submicromolar concentrations in isolated PTs from rats and humans. The rapid effects (within a few minutes) suggest that azPC activates NBCe1 and NHE via nongenomic signaling. The stimulatory effects were completely blocked by specific PPARγ antagonist GW9662, ERK kinase inhibitor PD98059, and CD36 inhibitor sulfosuccinimidyl oleate. Treatment with an siRNA against PPAR gamma completely blocked the stimulation of both NBCe1 and NHE by azPC. Moreover, azPC induced ERK phosphorylation in rat and human kidney cortex tissues, which were completely suppressed by GW9662 and PD98059 treatments. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via a CD36/PPARγ/mitogen-activated protein/ERK kinase/ERK pathway. We conclude that the stimulatory effects of azPC on PT transport may be partially involved in volume expansion.     

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-α in the primary cilium

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) α–mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-α and the Na⁺/H⁺ exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737^orpk MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-α ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5′-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA–mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737^orpk MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-α signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-α stimulation.     

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Kv7 K⁺-channel subunits differ in their apparent affinity for PIP₂ and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP₂ affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M₁ receptors by oxo-M leads to PIP₂ depletion through G_q and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M₁ receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC₅₀ for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhib_max) was 74 ± 3% (n = 5–7). When tonic PIP₂ abundance was increased by overexpression of PIP 5-kinase, the EC₅₀ was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhib_max (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3^T) that increases whole-cell currents by 30-fold without any change in apparent PIP₂ affinity. Kv7.3^T currents had a slightly right-shifted EC₅₀ as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhib_max (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhib_max was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP₂ by coexpressing them with Kv7.3^T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3^T) or higher affinity (Kv7.2 (R463E)/Kv7.3^T) channels, the EC₅₀ and Inhib_max were similar to Kv7.4 or Kv7.3^T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3^T. The resulting heteromer displayed a very low EC₅₀ for inhibition (32 ± 8 nM) and a slightly increased Inhib_max (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP₂ hydrolysis, PIP₂ binding to Kv7-channel subunits, and K⁺ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP₂ affinities.     

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis

AIM: To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis.METHODS: The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3^rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot.RESULTS: The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05).CONCLUSION: Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Long-Term Changes in Human Colonic Bifidobacterium Populations Induced by a 5-Day Oral Amoxicillin-Clavulanic Acid Treatment

The objective of this study was to assess the possible modifications due to amoxicillin-clavulanic acid (AMC) treatment on total bacteria and on Bifidobacterium species balance in human colonic microbiota. Eighteen healthy volunteers (19 to 36 years old) were given a 875/125 mg dose of AMC twice a day for 5 days. Fecal samples were obtained before and after antibiotic exposure. After total DNA extraction, total bacteria and bifidobacteria were specifically quantified using real-time PCR. Dominant species were monitored over time using bacterial and bifidobacterial Temporal Temperature Gradient gel Electrophoresis (TTGE). At the end of AMC exposure, total bacterial concentrations as well as bifidobacteria concentrations were significantly reduced compared to before AMC exposure:10.7±0.1 log₁₀ 16S rRNA gene copies/g vs 11.1±0.1 log₁₀ (p = 0.003) and 8.1±0.5 log₁₀ 16S rRNA gene copies/g vs 9.4±0.3 log₁₀ (p = 0.003), respectively. At the same time, the mean similarity percentages of TTGE bacteria and TTGE bifidobacteria profiles were significantly reduced compared to before AMC exposure: 51.6%±3.5% vs 81.4%±2.1% and 55.8%±7.6% vs 84.5%±4.1%, respectively. Occurrence of B. adolescentis, B. bifidum and B. pseudocatenulatum/B. catenulatum species significantly decreased. Occurrence of B. longum remained stable. Moreover, the number of distinct Bifidobacterium species per sample significantly decreased (1.5±0.3 vs 2.3±0.3; p = 0.01). Two months after AMC exposure, the mean similarity percentage of TTGE profiles was 55.6% for bacteria and 62.3% for bifidobacteria. These results clearly demonstrated that a common antibiotic treatment may qualitatively alter the colonic microbiota. Such modifications may have potential long-term physiological consequences.     

Resveratrol Attenuates the Na+-Dependent Intracellular Ca2+ Overload by Inhibiting H2O2-Induced Increase in Late Sodium Current in Ventricular Myocytes

Background/Aims

Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H₂O₂)-induced increase in late sodium current (I _Na.L) which augmented the reverse Na⁺-Ca²⁺ exchanger current (I _NCX), and the diastolic intracellular Ca²⁺ concentration in ventricular myocytes.

Methods

I _Na.L, I _NCX, L-type Ca²⁺ current (I _Ca.L) and intracellular Ca²⁺ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.

Results

Resveratrol (10, 20, 40 and 80 µM) decreased I _Na.L in myocytes both in the absence and presence of H₂O₂ (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), I _Na.L inhibitors, decreased I _Na.L in cardiomyocytes in the presence of 300 µM H₂O₂. H₂O₂ (300 µM) increased the reverse I _NCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H₂O₂ of the diastolic intracellular Ca²⁺ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca²⁺ transient rise and decay. As expected, 2 µM TTX had no effect on I _Ca.L.

Conclusion

Resveratrol protects the cardiomyocytes by inhibiting the H₂O₂-induced augmentation of I _Na.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.     

Ocular Dominance Is Associated with the Ganglion Cell-Inner Plexiform Layer Thickness Profile in the Macula

Purpose

To investigate the characteristics of macular ganglion cell-inner plexiform layer (GCIPL) thickness profiles associated with ocular dominance.

Setting

Private practice, Seoul, Republic of Korea.

Design

Comparative case-control study.

Methods

Both eyes of 199 participants with no ophthalmic abnormalities were included. Participants were imaged by spectral-domain optical coherence tomography, and underwent dominant eye testing using a hole-in-a-card test (sighting dominance) at the same visit. Macular GCIPL, as well as circumpapillary retinal nerve fiber layer (RNFL) thickness were compared for individual patients, according to ocular dominance.

Results

Ocular dominance occurred predominantly in the right eye (right vs. left: 72.36 vs. 27.60%; P < 0.001). In the comparison of macular GCIPL thickness, the average (81.27±5.01 μm vs. 80.66±6.31 μm in dominant vs. non-dominant eyes), inferonasal (81.39±5.47μm vs. 80.33±6.82μm, and inferior sectors (77.95±6.05μm vs. 76.97±8.15μm) were significantly different between dominant and non-dominant eyes (P = 0.040, 0.005, and 0.032, respectively). Significant predictors of average GCIPL thickness were spherical equivalent (β = 1.37, P<0.001), astigmatic power (β = 1.44, P = 0.009), disc area (β = 3.90, P < 0.001), average RNFL thickness (β = 0.22, P<0.001), average cup-to-disc ratio (β = 5.74, P = 0.002), difference between the inferior and superior quadrant RNFL thicknesses (β = 0.08, P = 0.024), and ocular dominance (β = 2.10, P = 0.020). On multivariate regression analysis, ocular dominance was correlated with average GCIPL thickness after adjusting for potential confounders (β = 1.63, P = 0.048).

Conclusions

Dominant eyes accompanied significantly thicker average macular GCIPL. This information suggests that macular GCIPL thickness may provide an indicator of the relative dominance of an eye.