Plant sterols cause macrothrombocytopenia in a mouse model of sitosterolemia

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5(-/-)) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5(-/-) mice displayed normal platelets, whereas Abcg5(-/-) mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5(-/-) mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment

Direct and indirect costs of tuberculosis among immigrant patients in the Netherlands

<p>Abstract</p> <p>Background</p> <p>In low tuberculosis (TB) incidence countries TB affects mostly immigrants in the productive age group. Little empirical information is available about direct and indirect TB-related costs that patients face in these high-income countries. We assessed the direct and indirect costs of immigrants with TB in the Netherlands.</p> <p>Methods</p> <p>A cross-sectional survey at 14 municipal health services and 2 specialized TB hospitals was conducted. Interviews were administered to first or second generation immigrants, 18 years or older, with pulmonary or extrapulmonary TB, who were on treatment for 1–6 months. Out of pocket expenditures and time loss, related to TB, was assessed for different phases of the current TB illness.</p> <p>Results</p> <p>In total 60 patients were interviewed. Average direct costs spent by households with a TB patient amounted €353. Most costs were spent when being hospitalized. Time loss (mean 81 days) was mainly due to hospitalization (19 days) and additional work days lost (60 days), and corresponded with a cost estimation of €2603.</p> <p>Conclusion</p> <p>Even in a country with a good health insurance system that covers medication and consultation costs, patients do have substantial extra expenditures. Furthermore, our patients lost on average 2.7 months of productive days. TB patients are economically vulnerable.</p

NiOx passivation in perovskite solar cells: from surface reactivity to device performance

Non-stoichiometric nickel oxide (NiOx) is the only metal oxide successfully used as hole transport material in p-i-n type perovskite solar cells (PSCs). Its favorable opto-electronic properties and facile large-scale preparation methods are potentially relevant for future commercialization of PSCs, though currently low operational stability of PSCs containing NiOx hole transport layers are reported. Poorly understood degradation reactions at the interface to the perovskite are seen as cause for the inferior stability and a variety of interface passivation approaches have been shown to be effective in improving the overall solar cell performance. To gain a better understanding of the processes happening at this interface, we systematically passivated possible specific defects on NiOx with three different categories of organic/inorganic compounds. The effects on the NiOx and the perovskite (MAPbI3) were investigated using x-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and scanning electron microscopy (SEM) where we find that the structural stability and film formation can be significantly affected. In combination with Density Functional Theory (DFT) calculations, a likely origin of NiOx-perovskite degradation interactions is proposed. The surface passivated NiOx was incorporated into MAPbI3 based PSCs and its influence on overall performance, particularly operational stability, was investigated by current-voltage (J-V), impedance spectroscopy (IS), and open circuit voltage decay (OCVD) measurements. Interestingly, we find that a superior structural stability due to an interface passivation must not relate to high operational stability. The discrepancy comes from the formation of excess ions at the interface which negatively impacts all solar cell parameters

Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells

As shown using clonal assays, the mouse HSC population undergoes quantitative as well as qualitative changes with age, including lineage differentiation, HSC pool size, marrow-homing efficiency, and self-renewal

ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway

ZFP36L1 negatively regulates erythroid differentiation of human hematopoietic progenitors by directly binding the 3′ UTR of Stat5b mRNA, thereby triggering its degradation. This study shows that posttranscriptional regulation is involved in the control of hematopoietic differentiation

Outcomes of elective liver surgery worldwide: a global, prospective, multicenter, cross-sectional study

Background: The outcomes of liver surgery worldwide remain unknown. The true population-based outcomes are likely different to those vastly reported that reflect the activity of highly specialized academic centers. The aim of this study was to measure the true worldwide practice of liver surgery and associated outcomes by recruiting from centers across the globe. The geographic distribution of liver surgery activity and complexity was also evaluated to further understand variations in outcomes. Methods: LiverGroup.org was an international, prospective, multicenter, cross-sectional study following the Global Surgery Collaborative Snapshot Research approach with a 3-month prospective, consecutive patient enrollment within January–December 2019. Each patient was followed up for 90 days postoperatively. All patients undergoing liver surgery at their respective centers were eligible for study inclusion. Basic demographics, patient and operation characteristics were collected. Morbidity was recorded according to the Clavien–Dindo Classification of Surgical Complications. Country-based and hospital-based data were collected, including the Human Development Index (HDI). (NCT03768141). Results: A total of 2159 patients were included from six continents. Surgery was performed for cancer in 1785 (83%) patients. Of all patients, 912 (42%) experienced a postoperative complication of any severity, while the major complication rate was 16% (341/2159). The overall 90-day mortality rate after liver surgery was 3.8% (82/2,159). The overall failure to rescue rate was 11% (82/ 722) ranging from 5 to 35% among the higher and lower HDI groups, respectively. Conclusions: This is the first to our knowledge global surgery study specifically designed and conducted for specialized liver surgery. The authors identified failure to rescue as a significant potentially modifiable factor for mortality after liver surgery, mostly related to lower Human Development Index countries. Members of the LiverGroup.org network could now work together to develop quality improvement collaboratives

Thrombopoietin-Mediated Activation of the mTOR/S6K/4E-BP Signaling Pathway Is Required for Proliferation in Megakaryocyte Progenitors.

Abstract Translational regulation plays a central role in cell proliferation, survival and cell differentiation through activation of the target of rapamycin (mTOR) signaling pathway. mTOR controls the phosphorylation status of proteins involved in initiating translational control, including ribosomal S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Recently, the mTOR and phosphoinositide 3-kinase (PI3-K) pathways have been linked through the tumor suppressor complex TSC1/2. The PI3-K target PKB inactivates the TSC1/2 complex which acts as a GAP for the Ras homologue Rheb to suppress mTOR signaling. As the regulation of cell number and cell size are important factors during megakaryopoiesis, we investigated the role of mTOR signaling in thrombopoietin (TPO) induced proliferation and differentiation in primary human megakaryocyte progenitors and in megakaryoblastic MO7e cells. Preincubation of isolated CD34+ cells, primary cultured CD61+ cells and MO7e cells with the mTOR inhibitor rapamycin (10 nM) resulted in highly specific inhibition of TPO-mediated 4E-BP1 (S65) phosphorylation, S6K (T389) phosphorylation and its substrate S6 (S235/236), without affecting PKB (S473) phosphorylation. Activation of the mTOR signaling pathway by TPO was dependent on the PI3-K pathway as LY294002 (10 μM) inhibited phosphorylation of 4E-BP1, S6K and S6. Treatment of MO7e cells with rapamycin inhibited TPO-induced proliferation and cell cycling by reducing cells in S-phase and blocking cells in G1. Rapamycin did not induce apoptosis as measured by cells in sub-G0 phase and by Annexin V expression. Suspension cultures of CD34+ cells treated with rapamycin resulted in a 2.3-fold reduction in overall cell proliferation (p=0.01) and a reduction in the percentage of CD61+ megakaryocytic cells (55.2±7.93 vs 35.0±5.42, p=0.08) generated after 7 days. The mean fluorescence intensity of CD61 and CD42 expressing cells was however not decreased. In addition, polyploidisation levels of the CD61 expressing cells cultured in the presence of rapamycin were not decreased, indicating rapamycin inhibited megakaryocyte proliferation, not differentiation. Further analysis revealed that transforming growth factor β1 (TGF-β1), which inhibits proliferation of megakaryocyte progenitors, downregulated TPO-induced S6K/S6 phosphorylation. Concluding, we have shown that the mTOR pathway is activated by TPO and plays a role in regulating proliferation in megakaryocyte progenitors. Part of the effect of the PI3-K/PKB and TGF-β pathways in regulating proliferation may be mediated by the mTOR/S6K/4E-BP1 signaling pathway.</jats:p

Effects of Age and Environment on Short-Term Homing and Function of Mouse Hematopoietic Stem Cells.

Abstract Abstract 1616 The C57BL/6 mouse is one of the best studied models of hematopoietic stem cell (HSC) aging. Characteristic age-related changes include an increase in HSC frequency, a corresponding decrease in functional activity, myeloid skewing, and decreased in vivo homing ability. Studies of age-dependent differences in homing have primarily relied upon the injection of young or old bone marrow into irradiated recipients, with subsequent quantification of functional homed HSCs using secondary transplantation assays (e.g. Liang et al, Blood 2005). While this is a relevant and informative approach, it is limited because it measures homed cells indirectly, using an assay that itself is dependent on homing. To study this phenomenon more directly, we compared the short-term homing efficiency of purified HSCs from old and young mice by co-injecting them into old or young recipients. This approach enabled us to ascertain whether the previously reported decrease in homing efficiency is due to differences in physical homing to the bone marrow, or a reduced function per homed cell, or some combination of both. Second, it allowed us to determine the relationship between the decreased in vivo homing efficiency of old HSCs and their functional properties when assayed in a homing-independent stem cell assay. Third, by varying the age and irradiation status of the homing recipients, we could test directly the influence of these factors on HSC homing and function. To accomplish these objectives, we purified HSCs (CD150+CD48-LSK bone marrow cells) from old (23-26 months) GFP+ and young (3-4 months) CFP+ mice, combined them at known ratios, and injected them into non-irradiated young, non-irradiated old, or lethally irradiated young recipients. 22 hours later, these recipients were sacrificed and the ratio of GFP+ and CFP+ cells found homed to the bone marrow were compared to the ratio in which they were injected. In this way, we determined that relative to their young counterparts, the 22-hour homing efficiency of old CD150+CD48-LSK cells was consistently 2-fold lower when injected into young recipients. However, when the same cells were injected into non-irradiated old recipients, the relative homing efficiency varied from 2-fold lower to 1.1-fold higher. This suggests that the factors affecting homing are similar between individual young recipients, while the microenvironments of individual old recipients are variable in their relative ability to support homing of young and old HSCs. We recently demonstrated that the proportion of purified old HSCs that were functional when assayed clonally in vitro (cobblestone area forming cell [CAFC] assay) declined approximately 2-fold compared to young HSCs. To determine whether the non-homed HSCs were the same cells that were non-functional in the CAFC assay, we compared the CAFC seeding efficiency of old and young cells before and after homing. There was no functional improvement in homed old LSK150+48- cells, suggesting that the homing defect and reduced CAFC efficiency are non-overlapping and may be characteristics of all old HSCs. To determine if the homing microenvironment also had an effect on the function of the homed cells, CAFC assays were initiated with old and young HSCs before and after homing into each of the three recipient types. This analysis revealed that the function of the homed cells was significantly decreased for age-mismatched or irradiated recipients compared to pre-homed cells. In contrast, CAFC activity of young or old cells homed to non-irradiated age-matched recipients did not change. This suggests that the environment of an age mismatched and/or irradiated recipient can have a negative effect on the function of homed cells within a matter of hours. In conclusion, these co-homing studies reveal that compared to young HSCs, old HSCs are physically less able to home and migrate into the bone marrow within a 22-hour period post-transplantation. However, this relative homing defect is most pronounced and consistent when injected into young recipients. Second, the homing defect and reduced CAFC efficiency are non-overlapping and may be characteristics of all old HSCs. Third, old or young HSCs that do successfully home to irradiated or age-mismatched recipients become functionally compromised compared to pre-homed HSCs, suggesting that the homing defects of old HSCs can be ameliorated or exacerbated depending on the microenvironment in which they are transplanted. Disclosures: No relevant conflicts of interest to declare. </jats:sec

STAT5 Is a Negative Regulator of Megakaryopoiesis.

Abstract The transcription factor STAT5 plays a critical role in self-renewal and lineage commitment of hematopoietic stem cels (HSCs). We have recently shown in CB CD34+ cells that persistent activation of STAT5 results in enhanced self-renewal and induces erythroid differentiation, while myelopoiesis was severely impaired. In this study we analyzed the function of STAT5 during megakaryocyte (MK) differentiation. Using RNA interference we downregulated STAT5 expression in peripheral blood CD34+ cells. Cells were transduced with a lentiviral construct encoding eGFP and a short-hairpin RNA for STAT5 or with a control vector expressing YFP. Transduction efficiencies were determined by flow cytometric detection of eGFP/YFP and ranged from 40%–80%. Decreased STAT5 expression was verified by Western blot and quantitative RT-PCR (65% lower expression in STAT5 RNAi cells versus YFP+ control cells, p=&amp;lt;0.01). To assess the effects of decreased STAT5 expression on the progenitor pool, the transduced populations were sorted and enumerated in colony assays. Downregulation of STAT5 significantly increased the number of MK progenitors (1.9-fold, p=0.02) and resulted in a decrease of erythroid progenitors (0.6-fold, p&amp;lt;0.01), but did not affect the number of granulocyte/monocyte progenitors. Prospective isolation of immature and commited progenitors are currently being performed to distinguish if certain progenitor subsets are re-directed to the MK lineage by low STAT5 expression. Next, we analyzed the role of STAT5 during MK differentiation in unilineage suspension cultures. Transduced cells were cultured in serum-free medium containing TPO and SCF and scored for cell counts and expression of MK markers weekly. The percentage of transduced cells and their number did not differ when STAT5 RNAi cells were compared to controls, indicating that downregulation of STAT5 provides no proliferative advantage or disadvantage. However, the expression of glycoproteins IIb/IIIa (CD41) and Ib (CD42) was upregulated, and an increase in large, polyploid cells was observed in STAT5 RNAi cultures (15.2% polyploid cells versus 4.9% in control cells at day 7, p&amp;lt;0.01). RT-PCR analysis of transcription factors predominantly expressed in MK and erythroid lineages revealed that Runx1 and Erg were increased in STAT5 RNAi cells, and the level of Scl was decreased compared to control cells. These observations correlate with the increased MK differentiation observed in STAT5 downregulated cells. Together, these data demonstrate that the expression level of STAT5 has important effects regarding the type of hematopoietic cell development, with high levels favouring erythroid development and low levels enhancing MK differentiation and maturation.</jats:p