Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

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Potassium effects on NCC are attenuated during inhibition of Cullin E3-ubiquitin ligases

10.1101/2021.11.30.470531 ◽

2021 ◽

Author(s):

Sathish K Murali ◽

Robert Little ◽

Soren B Poulsen ◽

Mohammed Ferdaus ◽

David H Ellison ◽

...

Keyword(s):

Sodium Chloride ◽

Ex Vivo ◽

Active Form ◽

Inverse Correlation ◽

Vital Role ◽

Renal Tubules ◽

Short Term ◽

Cullin 3 ◽

K Concentration

The thiazide sensitive sodium-chloride co-transporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by the with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which are controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the Cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of Cullins (Cul1, 3, 4 and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect Cullin neddylation levels in ex vivo renal tubules. Short-term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan Cullin inhibitor), but the response to low extracellular K+ was absent. Long-term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other Cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

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A Novel LSTM Model with Interaction Dual Attention for Radar Echo Extrapolation

Remote Sensing ◽

10.3390/rs13020164 ◽

2021 ◽

Vol 13 (2) ◽

pp. 164

Author(s):

Chuyao Luo ◽

Xutao Li ◽

Yongliang Wen ◽

Yunming Ye ◽

Xiaofeng Zhang

Keyword(s):

Short Term Memory ◽

Weather Forecast ◽

Vital Role ◽

Data Sets ◽

Short Term ◽

Learning Techniques ◽

Radar Echo ◽

Hidden States ◽

Better Than

The task of precipitation nowcasting is significant in the operational weather forecast. The radar echo map extrapolation plays a vital role in this task. Recently, deep learning techniques such as Convolutional Recurrent Neural Network (ConvRNN) models have been designed to solve the task. These models, albeit performing much better than conventional optical flow based approaches, suffer from a common problem of underestimating the high echo value parts. The drawback is fatal to precipitation nowcasting, as the parts often lead to heavy rains that may cause natural disasters. In this paper, we propose a novel interaction dual attention long short-term memory (IDA-LSTM) model to address the drawback. In the method, an interaction framework is developed for the ConvRNN unit to fully exploit the short-term context information by constructing a serial of coupled convolutions on the input and hidden states. Moreover, a dual attention mechanism on channels and positions is developed to recall the forgotten information in the long term. Comprehensive experiments have been conducted on CIKM AnalytiCup 2017 data sets, and the results show the effectiveness of the IDA-LSTM in addressing the underestimation drawback. The extrapolation performance of IDA-LSTM is superior to that of the state-of-the-art methods.

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Glucocorticoids Decrease the Conversion of Thyroxine into 3,5,3′-Tri-Iodothyronine by Isolated Rat Renal Tubules

Clinical Science ◽

10.1042/cs0620215 ◽

1982 ◽

Vol 62 (2) ◽

pp. 215-220 ◽

Cited By ~ 5

Author(s):

P. Heyma ◽

R. G. Larkins

Keyword(s):

Strong Evidence ◽

Glucocorticoid Receptors ◽

Actinomycin D ◽

Time Dependent ◽

Renal Tubules ◽

Short Term ◽

Collagenase Digestion ◽

Isolated Rat

1. The effect of glucocorticoids on the deiodination of thyroxine (T4) to 3,5,3′-tri-iodothyronine (T3) was studied in rat renal tubules prepared by collagenase digestion. 2. In short-term (6 h) experiments, cortisol and dexamethasone inhibited the conversion of T4 into T3 at concentrations of 2 × 10-4 mol/l and 2 × 10-5 mol/l respectively. The inhibition by cortisol and dexamethasone was time dependent and was prevented by actinomycin D and progesterone, suggesting that the inhibition is mediated by an effect on nuclear transcription dependent on binding to glucocorticoid receptors. 3. In long-term (16 h) experiments, cortisol and dexamethasone inhibited T4 to T3 conversion by the tubules at concentrations of 1 × 10-12 mol/l and above. In addition, physiological concentrations of corticosterone (1 × 10-8 mol/l) were able to decrease T3 generation from T4. 4. Our data provide strong evidence that physiological concentrations of glucocorticoids are able to affect T3 production from T4 directly and suggest that they may be important regulators of T4 deiodination.

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Nicotinamide, a Recognized Inhibitor of SIRT1, Promotes Expansion in Ex Vivo Cultures of Short and Long-Term Repopulating Cells.

Blood ◽

10.1182/blood.v114.22.2431.2431 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2431-2431 ◽

Cited By ~ 1

Author(s):

Tony Peled ◽

Hadas Shoham ◽

Dorit Aschengrau ◽

Dima Yackoubov ◽

Gabi Frei ◽

...

Keyword(s):

Peripheral Blood ◽

Cultured Cells ◽

Ex Vivo ◽

Donor Cell ◽

Mice Model ◽

Short Term ◽

Post Transplantation ◽

Short And Long Term ◽

Cells Cultured

Abstract Abstract 2431 Poster Board II-408 Nicotinamide (NAM), is a form of VitB3 that recognized and inhibits SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase. We have previously demonstrated that NAM inhibits in vitro differentiation and enhances expansion, migration, homing and NOD/SCID engraftment efficacy of cord blood (CB)-derived CD34+ cells cultured with cytokines. In the current study, the in vivo function of ex vivo cultured cells with NAM was tested in a congenic mice model (BALB/C, CD45.1/CD45.2) for BM transplantation. Purified CD117+ BM cells from BALB/C CD45.1 mice were cultured with a combination of 4 cytokines (FLT3, SCF, TPO, IL-6, 50 ng/ml each), with and without 0.5mM NAM for three weeks. Numbers of CFUc, CD117+ and CD117+Lin- cells were significantly (p < 0.05) higher in cultures treated with NAM as compared with cultured treated with cytokines alone. Non-cultured, freshly purified CD117+ cells (1000 and 2500 cells/mice) and their total progeny following expansion with or without NAM were transplanted into ablated (1000 Rad) CD45.2 mice (n = 10/cohort), 24h post irradiation (Fig 1). Three months post transplantation, all the mice in the control group (non-transplanted) died. The percent survival of mice transplanted with cells cultured with cytokines and NAM was remarkably higher over the survival of mice in the cohort transplanted with cells cultured with cytokines alone or non-cultured cells (Fig 1). FACS analysis (CD45.1-donor / CD45.2-host) of peripheral blood from mice transplanted with NAM cultured cells show 80% donor cell chimerism (CD45.1), 3 and 6 months post transplantation. Percent of donor derived Gr-1+ and CD3+ cells were similar in mice transplanted with non-cultured or NAM cultured cells. Percentages of donor cell chimerism (CD45.1) in secondary mice transplanted with total BM cells derived from primary recipients originally transplanted with non-cultured and NAM cultured cells were 47 and 73, respectively, 6 weeks after the secondary transplantation. In a different experiment, to follow time to engraftment during the first month post transplantation, mice transplanted with non-cultured cells or cells cultured with cytokines and NAM (n = 10/cohort) were bled at weekly intervals and peripheral blood samples were counted for WBC and analyzed by the FACS to determine donor cell chimerism and lineage engraftment. The results show accelerated engraftment (Fig 2) and higher levels of donor cell chimerism (Fig 3) in the cohort transplanted with NAM cultured cells relative to the cohort transplanted with non-cultured cells. Number of granulocytes, T, NK and B cells during the first month post transplant were also significantly (p<0.05) higher in mice transplanted with cells cultured with cytokines and NAM relative to their levels in mice transplanted with non-cultured cells. The results obtained in the congenic mice model for BMT suggest that NAM promotes expansion in ex vivo cultures of short and long-term repopulating cells, as demonstrated by accelerated donor derived engraftment during the first month post transplantation, higher survival of mice, sustained donor cell chimerism 6 month post transplantation and successful reconstitution of secondary recipients. NAM is thus a novel molecule that may be used to stimulate and expand hematopoietic repopulating cells, fasten post transplant engraftment and hopefully improve transplantation outcome. Current studies are designed to elucidate NAM mode of action. Fig 1: Three month survival Fig 1:. Three month survival Fig 2: Short-term Engrafoment Fig: 3 Percentage of Donor Chimerism Fig 2:. Short-term Engrafoment Fig: 3 Percentage of Donor Chimerism Disclosures: Peled: Gamida-Cell: Employment, Equity Ownership. Shoham:Gamida Cell: Employment. Aschengrau:Gamida Cell: Employment. Yackoubov:Gamida Cell: Employment. Frei:Gamida Cell: Employment. Nagler:Gamida Cell: Arnon Nagler, Consultancy. Peled:Gamida Cell: Consultancy.

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Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long- term repopulating stem cells

Blood ◽

10.1182/blood.v88.9.3642.bloodjournal8893642 ◽

1996 ◽

Vol 88 (9) ◽

pp. 3642-3653 ◽

Cited By ~ 41

Author(s):

SJ Szilvassy ◽

KP Weller ◽

B Chen ◽

CA Juttner ◽

A Tsukamoto ◽

...

Keyword(s):

Stem Cells ◽

Blood Cells ◽

Ex Vivo ◽

Granulocyte Colony Stimulating Factor ◽

Short Term ◽

Hematopoietic Stem ◽

Colony Forming Units ◽

Sustained Recovery ◽

Stimulating Factor

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca- 1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.

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Notch2 Signaling Inhibits Differentiation and Promotes Self Renewal of Hematopoietic Stem and Progenitor Cells During Marrow Regeneration.

Blood ◽

10.1182/blood.v114.22.2544.2544 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2544-2544

Author(s):

Barbara Varnum-Finney ◽

Irwin D. Bernstein

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Myeloid Differentiation ◽

Primary Control ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem ◽

Post Transplant

Abstract Abstract 2544 Poster Board II-521 Notch regulates numerous lineage choices during vertebrate development, and although ex vivo studies suggest that Notch regulates hematopoietic stem cell (HSC) and multipotential progenitor (MPP) differentiation, a functional role for Notch in HSC/MPP self renewal in vivo remains controversial. We previously reported a Notch2 signaling role during bone marrow (BM) recovery following injection with chemotherapeutic agent 5-fluorouracil (5FU), where Notch2 signaling impedes myeloid differentiation, allowing for generation of sufficient numbers of progenitor cells. Herein, we examine a Notch2 signaling role in HSC as well as progenitor cell self renewal by enumerating generation of HSC and short term repopulating cells in lethally irradiated recipients (Ly5.1+) transplanted with a limiting number (5 × 105) of BM cells from either control mice or from mice bearing Cre-LoxP-inducible Notch2 deletions (Ly5.2+). In recipient mice transplanted with control BM, recovery was evident from Day11 to Day13 post transplant when significantly more than the initial post-irradiation number of 9.0 × 106 BM cells was seen in the recovering marrow. In recovering mice, recipients receiving control cells generated more BM cells than did recipients receiving Notch2-deficient cells. Furthermore, mice receiving control cells generated significantly more donor Sca-1+c-kit+ (SK+) cells than recipients receiving Notch2-deficient BM cells [44.4×103 (s.e.m.+/− 14×103) vs 8.2×103 (s.e.m.+/−1.5×103), respectively, p=0.001]. To quantitate the generation of short term repopulating cells, secondary radioprotection assays were performed. Irradiated secondary recipient mice received 1×106 BM cells from the primary recipients previously transplanted with either control cells or Notch2-deficient cells. Secondary recipients receiving cells from primary control transplants survived significantly longer than those receiving cells from primary Notch2-deficient transplants or than irradiated mice receiving no cells (n=4, p=0.01), indicating Notch2 is required to generate sufficient numbers of cells to provide radioprotection. To quantitate long term HSC generated in the recovering marrow, competitive repopulating units (CRU) were enumerated by performing secondary transplants in which 4-doses of BM cells ranging from 4 × 104 to 5 × 106 cells from primary transplants were injected into secondary recipients along with 1 × 105 Ly5.1+ competing cells. Enumeration of CRU at 2 weeks post transplant confirmed the number of short term repopulating cells was significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(1.3 CRU vs 8.8 CRU / 1×106 BM cells, respectively), p=0.0004)]. Enumeration of CRU at 9 weeks post transplant indicated HSC numbers were also significantly decreased in mice transplanted with Notch2-deficient cells compared to mice transplanted with control cells [(0.1 CRU vs 0.7 CRU / 1×106 BM cells, respectively), p=0.02]. Taken together, our results demonstrate a role for Notch2 in enhancing generation of long term HSC as well as short term repopulating cells and suggests that Notch2 signaling regulates a hierarchy of events to assure the initial repopulation by HSC and MPP, while delaying myeloid differentiation during hematopoietic regeneration. Disclosures: No relevant conflicts of interest to declare.

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An Ex Vivo Investigation into the Release of Fluoride from Fluoride-containing Orthodontic Bonding Composites

British Journal of Orthodontics ◽

10.1179/bjo.21.3.239 ◽

1994 ◽

Vol 21 (3) ◽

pp. 239-243 ◽

Cited By ~ 9

Author(s):

Sarah H. A. Ghani ◽

Stephen L. Creanor ◽

John K. Luffingham ◽

Richard H. Foye

Keyword(s):

Similar Pattern ◽

Ex Vivo ◽

Composite Resins ◽

Fluoride Release ◽

Short Term ◽

Ex Vivo Model ◽

The Third ◽

Orthodontic Bonding

This study was concerned with an evaluation of fluoride release from commercially available orthodontic bonding composite resins, known as Reliance® and Mirage Dual Cure®, which are claimed to release ionic floride. Forty-eight premolar teeth had brackets bonded with four different composite resins—Mirage Dual Cure®, Reliance®, Right-on® and Heliosit®. They were then immersed in a demineralizing solution. The amount of fluoride released from the composites into the solution was measured. The results indicated that Mirage Dual Curereg; released statistically significant amounts of fluoride over the first 2 days. A similar pattern was noted with Reliance® albeit releasing a lesser amount. From the third day onwards, fluoride release levelled out to concentrations similar to those of the two control materials, Right-on® and Heliosit®(i.e. 0·09 ppm). Fluoride-releasing composite resins, therefore, failed to demonstrate any potential long-term fluoride release within the ex vivo model. Even in the short term, the amount of fluoride released was very small.

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Non-invasive monitoring of pH and oxygen using miniaturized electrochemical sensors in an animal model of acute hypoxia

Journal of Translational Medicine ◽

10.1186/s12967-021-02715-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Laura Pla ◽

Sergio Berdún ◽

Mònica Mir ◽

Lourders Rivas ◽

Sandrine Miserere ◽

...

Keyword(s):

Continuous Monitoring ◽

Electrochemical Sensors ◽

Ex Vivo ◽

Acute Hypoxia ◽

Fetal Hypoxia ◽

Short Term ◽

Short Term Evaluation ◽

Term Evaluation

Abstract Background One of the most prevalent causes of fetal hypoxia leading to stillbirth is placental insufficiency. Hemodynamic changes evaluated with Doppler ultrasound have been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. As a first step, the present work aimed to evaluate the performance of miniaturized electrochemical sensors in the continuous monitoring of oxygen and pH changes in a model of acute hypoxia-acidosis. Methods pH and oxygen electrochemical sensors were evaluated in a ventilatory hypoxia rabbit model. The ventilator hypoxia protocol included 3 differential phases: basal (100% FiO2), the hypoxia-acidosis period (10% FiO2) and recovery (100% FiO2). Sensors were tested in blood tissue (ex vivo sensing) and in muscular tissue (in vivo sensing). pH electrochemical and oxygen sensors were evaluated on the day of insertion (short-term evaluation) and pH electrochemical sensors were also tested after 5 days of insertion (long-term evaluation). pH and oxygen sensing were registered throughout the ventilatory hypoxia protocol (basal, hypoxia-acidosis, and recovery) and were compared with blood gas metabolites results from carotid artery catheterization (obtained with the EPOC blood analyzer). Finally, histological assessment was performed on the sensor insertion site. One-way ANOVA was used for the analysis of the evolution of acid-based metabolites and electrochemical sensor signaling results; a t-test was used for pre- and post-calibration analyses; and chi-square analyses for categorical variables. Results At the short-term evaluation, both the pH and oxygen electrochemical sensors distinguished the basal and hypoxia-acidosis periods in both the in vivo and ex vivo sensing. However, only the ex vivo sensing detected the recovery period. In the long-term evaluation, the pH electrochemical sensor signal seemed to lose sensibility. Finally, histological assessment revealed no signs of alteration on the day of evaluation (short-term), whereas in the long-term evaluation a sub-acute inflammatory reaction adjacent to the implantation site was detected. Conclusions Miniaturized electrochemical sensors represent a new generation of tools for the continuous monitoring of hypoxia-acidosis, which is especially indicated in high-risk pregnancies. Further studies including more tissue-compatible material would be required in order to improve long-term electrochemical sensing.

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Non-Invasive Monitoring of pH and Oxygen Using Miniaturized Electrochemical Sensors in an Animal Model of Acute Hypoxia

10.21203/rs.3.rs-95118/v1 ◽

2020 ◽

Author(s):

Laura Pla Codina ◽

Sergio Berdún ◽

Mònica Mir ◽

Lourders Rivas ◽

Sandrine Miserere ◽

...

Keyword(s):

Continuous Monitoring ◽

Electrochemical Sensors ◽

Ex Vivo ◽

Acute Hypoxia ◽

Fetal Hypoxia ◽

Short Term ◽

Short Term Evaluation ◽

Term Evaluation

Abstract BackgroundOne of the most prevalent causes of fetal hypoxia leading to stillbirth is placental insufficiency. Hemodynamic changes evaluated with Doppler ultrasound have been used as a surrogate marker of fetal hypoxia. However, Doppler evaluation cannot be performed continuously. As a first step, the present work aimed to evaluate the performance of miniaturized electrochemical sensors in continuous monitoring of oxygen and pH changes in a model of acute hypoxia-acidosis. MethodspH and oxygen electrochemical sensors were evaluated in a ventilatory hypoxia rabbit model. The ventilator hypoxia protocol included three differential phases: basal (100% FiO2), hypoxia-acidosis period (10% FiO2) and recovery (100% FiO2). Sensors were tested in blood tissue (ex vivo sensing) and in the muscular tissue (in vivo sensing). pH electrochemical and oxygen sensors were evaluated at the same day of insertion (short-term evaluation) and pH electrochemical sensors were also tested after 5 days of insertion (long-term evaluation). pH and oxygen sensing were registered during all the ventilatory hypoxia protocol (basal, hypoxia-acidosis and recovery) and were compared with blood gas metabolites results from carotid artery catheterization (EPOC® blood analyzer). Finally, histological assessment was performed on the site of the sensor’s insertion. One-way ANOVA was used for the analysis of the evolution of acid-based metabolites and electrochemical sensor signaling results; T-test was used for pre and post calibration analyses; and chi-square analyses for categorical variables. ResultsAt the short-term evaluation, both pH and oxygen electrochemical sensors distinguished the basal and hypoxia-acidosis periods in the in vivo and ex vivo sensing. However, only the ex vivo sensing detected recovery period. At the long-term evaluation, pH electromechanical sensor signal seemed to lose sensibility. Finally, histological assessment revealed no signs of alteration at the same day of evaluation (short-term), whereas at the long-term evaluation sub-acute inflammatory reaction adjacent to the site of the implantation was detected. ConclusionsThe use of miniaturized electrochemical sensors open a new generation of tools for continuous monitoring of hypoxia-acidosis, especially indicated in high risk pregnancies. Further studies including more tissue-compatible material would be required in order to improve the long-term electromechanical sensing.

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Integrin-α3 Is a New Marker of Long-Term Hematopoietic Stem Cells in Ex Vivo Expanded Cells

Blood ◽

10.1182/blood-2018-99-111963 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1267-1267

Author(s):

Elisa Tomellini ◽

Iman Fares ◽

Bernhard Lehnertz ◽

Jalila Chagraoui ◽

Nadine Mayotte ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stem Cell Markers ◽

Short Term ◽

Differentiation Potential ◽

Hematopoietic Stem

Abstract Hematopoietic stem cell (HSC) transplantation constitutes one of the most effective therapeutic strategies to treat numerous hematological diseases. Cord blood (CB) is one of the most attractive donor sources of stem cells for this procedure due to its rapid availability, HLA mismatches tolerance and low associated risk of chronic graft-versus-host disease. However, these advantages are offset by the limited cell dose in CB units, which can contribute to delayed hematopoietic engraftment following transplantation. Mastering ex vivo HSC expansion is therefore of great interest for clinical purposes and for genetic manipulation. HSCs can be functionally defined as either long-term (LT-HSC), providing life-long hematopoiesis and characterized by delayed engraftment pattern, or short-term repopulating stem cells (ST-HSC), providing early and transient hematopoietic recovery. Major hurdles hindering the study of these cell populations is the current inability to evaluate their content in cultured samples and the lack of understanding of the molecular mechanisms regulating stem cell self-renewal ex vivo. Those issues highly benefited from the discovery by our laboratory of the small molecule UM171, which promote HSC expansion ex vivo, as well as from the identification of EPCR as one of the most reliable surface markers for cultured HSCs. We now describe the identification of Integrin-α3 (ITGA3) as a novel marker for cultured HSCs. ITGA3 expression was found to be sufficient to split the primitive EPCR+CD90+CD133+CD34+CD45RA- HSC population in two functionally distinct fractions presenting only short-term (ITGA3-) and both short-term and long-term (ITGA3+) repopulating potential. ITGA3+ cells, as opposed to the ITGA3- fraction, exhibited robust multilineage differentiation potential and serial reconstitution ability in immunocompromised mice. This combination of markers identifies repopulating HSCs in culture by FACS beyond what is currently possible with other approaches, with a frequency of LT-HSC found in the ITGA3+ population estimated at 1:38 in day 7 UM171 expanded CB-cells. Moreover, lentiviral-mediated ITGA3 knockdown was shown to compromise the LT repopulating activity of cultured HSC in vivo. Gene expression profiling revealed striking molecular similarity between ITGA3+ and ITGA3- cells, showing overrepresentation of genes involved in fundamental hematopoietic programs known to govern HSC specification and function in both of these populations. However, ITGA3+ and ITGA3- subsets clearly clustered separately by principle component analysis, indicating broad differences in gene expression. Concordantly with their primitive phenotype, stem cell markers and cell quiescence are gene sets enriched in ITGA3+ cells, while progenitor markers, DNA replication, M/G1 and G2/M checkpoints, mRNA processing, reduction of hypoxia and Myc targets were significantly downregulated in these cells. Altogether, our results indicate that ITGA3 is a reliable marker for cultured HSCs, improving the accuracy of prospective HSC identification in culture. Deciphering the function of genes upregulated in primitive ITGA3+ HSCs will represent an invaluable resource for dissecting the genetic programs that govern hematopoietic stem cells biology. Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

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