Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia.

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Subcutaneous progesterone versus vaginal progesterone for luteal phase support in in vitro fertilization: A retrospective analysis from daily clinical practice.

OBJECTIVE: Progesterone application for luteal phase support is a well-established concept in in vitro fertilization (IVF) treatment. Water-soluble subcutaneous progesterone injections have shown pregnancy rates equivalent to those observed in patients receiving vaginal administration in randomized controlled trials. Our study aimed to investigate whether the results from those pivotal trials could be reproduced in daily clinical practice in an unselected patient population. METHODS: In this retrospective cohort study in non-standardized daily clinical practice, we compared 273 IVF cycles from 195 women undergoing IVF at our center for luteal phase support with vaginal administration of 200 mg of micronized progesterone three times daily or subcutaneous injection of 25 mg of progesterone per day. RESULTS: Various patient characteristics including age, weight, height, number of oocytes, and body mass index were similar between both groups. We observed no significant differences in the clinical pregnancy rate (CPR) per treatment cycle between the subcutaneous (39.9%) and vaginal group (36.5%) (p=0.630). Covariate analysis showed significant correlations of the number of transferred embryos and the total dosage of stimulation medication with the CPR. However, after adjustment of the CPR for these covariates using a regression model, no significant difference was observed between the two groups (odds ratio, 0.956; 95% confidence interval, 0.152-1.786; p=0.888). CONCLUSION: In agreement with randomized controlled trials in study populations with strict selection criteria, our study determined that subcutaneous progesterone was equally effective as vaginally applied progesterone in daily clinical practice in an unselected patient population.

The function of Chalcogenophene in the Cyclomatelated Ring of the Cycloruthenated Dyes applied in Dye-Sensitized Solar Cell.

Three thiocyanate-free cycloruthenated complexes, DUY24-O, DUY24, and DUY24-Se containing furan, thiophene, and selenophene, respectively, as a part of the cycloruthenated ring, were designed to reveal the function of the chalcogen atom on the physicochemical and photovoltaic performance of the cycloruthenated sensitizers applied in dye-sensitized solar cells (DSCs). The three sensitizers have a similar molecular size; therefore, the effect of molecular dimensions on their photovoltaic performance can be negligible. NMR data, electron-donating resonance effects, optical properties, and the energy levels of the frontier orbitals reveal that the physical/photovoltaic properties of the three sensitizers were affected significantly by the chalcogen atom on the cyclometalated chalcogenophene ring. The λmax (both in ethanol and adsorbed on TiO2), frontier orbital level, and dye loading of thiophene- and selenophene-containing dyes are very close. Nevertheless, DUY24-Se has a higher molar absorption coefficient compared to DUY24; therefore, the DSC based on DUY24-Se has higher efficiency (8.4% under AM1.5 G one-sun and 26% under T5-light at ca. 6000 lux) than that sensitized with the DUY24 dye. These efficiencies are also higher than those (7.9 and 21.6%, respectively) of the cell dyed with N719, fabricated using the same conditions. The better performance of the device sensitized with DUY24-Se compared to DUY24-based cells suggests that selenophene is as good as (or even better than) thiophene to be a part of the cyclometalated ring for thiocyanate-free cycloruthenated sensitizers applied in DSCs. Furan-containing DUY24-O has much worse photovoltaic performance compared to the other two dyes. This is not only because DUY24-O has the shortest λmax, the lowest molar absorption coefficient, and the highest HOMO level but also the lowest dye loading (because of the strong interaction between the oxygen in furan and TiO2, the array of DUY24-O occupies more surface when adsorbed on TiO2) and the fastest charge recombination. The physicochemical and photovoltaic properties as well as the adsorption behavior of the dye on the TiO2 anode for the cycloruthenated sensitizers affected significantly by the chalcogen atom of the chalcogenophene on the cyclometalated ring provide a new strategy to design high-efficiency NCS-free cyclometalated sensitizers for DSCs.

A new hopane derivative from the lichen Dirinaria applanata.

Chemical investigation of the lichen Dirinaria applanata led to isolate nine compounds including a new hopane derivative, 1ß-acetoxy-21α-hopane-3ß,22-diol (1) together with six phenolic compounds naming divaricatinic acid (2), methyl divaricatinate (3), methyl-ß-orcinolcarboxylate (4), methyl haematommate (5), divarinol (6), ramalinic acid A (7), and two xanthones namely lichenxanthone (8), 4,5-dichlorolichenxanthone (9). Their structures were elucidated by spectroscopic data in combination with published literature. Except compound 2, all compounds were isolated from this species for the first time.

Function of Tetrabutylammonium on High-Efficiency Ruthenium Sensitizers for Both Outdoor and Indoor DSC Application.

The function of tetrabutyl ammonium ions (TBA+) in a sensitizer used in dye-sensitized solar cells (DSC) is contradictory. TBA+ can reduce unwanted charge-recombination by protecting the TiO2 surface and reduce dye aggregation, enhancing the photovoltaic performance. It will also compete with the dye-loading on the TiO2 film, decreasing the short-circuit current density of the cell. Three ruthenium sensitizers (DYE III, DUY11, and DUY12 containing two H+, one H+/one TBA+, and two TBA+, respectively) were prepared to systematically investigate the function of TBA+ in a dye for DSC under both standard sunlight and indoor illumination. The optical properties and frontier orbital energy level of the sensitizers are not influenced significantly by the number of TBA+. Under the standard 1 sun illumination, DSCs based on DUY11 (containing one H+ and one TBA+) achieved the highest power conversion efficiency (PCE) of 11.47%. Overall, optimized DSCs sensitized by the three ruthenium dyes all have the PCE over 10%, which is higher than that (9.95%) of N719-dyed cell fabricated at the same conditions. Under the illumination of a light emitting diode (LED), DSCs sensitized by DUY11 also have the highest efficiency of 19%. Furthermore, DUY12 with two TBA+ exhibits superior photovoltaic performance compared to a DYE III (containing two H+ in the anchoring ligands)-dyed cell; although these two dyes have similar photovoltaic performance under standard 1 sun lighting. The important function of TBA+ in reducing the charge recombination (by protecting TiO2 surface and avoiding dye aggregation) of a DSC under indoor lighting (when small number of electrons were excited by weak light) is also revealed.

Loss of the selective autophagy receptor p62 impairs murine myeloid leukemia progression and mitophagy.

Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

High-Efficiency Cycloruthenated Sensitizers for Dye-Sensitized Solar Cells.

Four thiocyanate-free ruthenium sensitizers (DUY24-DUY27) containing 2-thienylpyridine moiety as cyclometalating core were synthesized for dye-sensitized solar cell (DSC) application. To the best of our knowledge, DUY24-DUY27 are four best-efficiency sensitizers having 42%-65% higher efficiencies compared to those of the published sensitizers comprising the same type of the cyclometalating ancillary ligands. The significant characteristic of DUY24-DUY27 is their ß-lowest unoccupied spin orbital (ß-LUSO) distributes remarkably on the cyclometalating ligands, especially on the soft sulfur atom, which strengthens the interaction between the oxidized dye and iodide ion for efficient dye regeneration. The photovoltaic performance of DUY24-DUY27-based DSCs supports that the dye regeneration (therefore the short-circuit photocurrent density (JSC) of the cell) can be improved by not only lowering the highest occupied molecular orbital energy level of the dye molecule but also distributing the ß-LUSO properly on the soft atoms. The study provides an important new guide for designing high-efficiency ruthenium-based dyes for DSC application.

Effect of the CF3 Substituents on the Charge-Transfer Kinetics of High-Efficiency Cyclometalated Ruthenium Sensitizers.

Six thiocyanate-free complexes, DUY1-DUY6, were synthesized, and their application in a dye-sensitized solar cell was studied to explore the effect of the CF3 substituent positioned in the ancillary ligand and the structure of the anchoring ligand on the physicochemical properties, charge-transfer kinetics, and photovoltaic properties of ruthenium sensitizers. When the electron-withdrawing groups were installed on the cyclometalating ligands and their π conjugation of the ancillary ligand was extended, the frontier orbital energy levels of the ruthenium complex appeared to be sufficient for effective electron injection and dye regeneration, at the same time having high light-harvesting ability. Two electron-withdrawing CF3 groups meta to the cyclometalated position reduce the electron density at the metal center less seriously than o-CF3 and p-CF3 groups. The sensitizers containing a m-CF3 group also reveal a more favorable distribution of ß lowest unoccupied spin orbital for interaction between the oxidized dyes and the iodide ion, which promotes dye regeneration. The absorption profiles of DUY1-DUY4 adsorbed a TiO2 film extended to longer wavelength compared to those in an N,N-dimethylformamide solution, especially DUY1 and DUY2 dyes, which have λmax red shifts of up to 30 nm. The DUY2-dyed cell exhibited the highest efficiency of 9.03%, while the power conversion efficiencies of DUY1-, DUY3-, DUY4-, and N719-based devices were 7.40%, 7.01%, 8.92%, and 8.63%, respectively. DUY5 and DUY6 (the side products of DUY3 and DUY4) without anchoring groups have very weak physical adsorption on a TiO2 anode. The corresponding cells exhibit very low efficiency (<0.1%), although both dyes have high light-harvesting ability and proper frontier orbital energy levels.

Maintenance of the enteric stem cell niche by bacterial lipopolysaccharides? Evidence and perspectives.

The enteric nervous system (ENS) has to respond to continuously changing microenvironmental challenges within the gut and is therefore dependent on a neural stem cell niche to keep the ENS functional throughout life. In this study, we hypothesize that this stem cell niche is also affected during inflammation and therefore investigated lipopolysaccharides (LPS) effects on enteric neural stem/progenitor cells (NSPCs). NSPCs were derived from the ENS and cultured under the influence of different LPS concentrations. LPS effects upon proliferation and differentiation of enteric NSPC cultures were assessed using immunochemistry, flow cytometry, western blot, Multiplex ELISA and real-time PCR. LPS enhances the proliferation of enteric NSPCs in a dose-dependent manner. It delays and modifies the differentiation of these cells. The expression of the LPS receptor toll-like receptor 4 on NSPCs could be demonstrated. Moreover, LPS induces the secretion of several cytokines. Flow cytometry data gives evidence for individual subgroups within the NSPC population. ENS-derived NSPCs respond to LPS in maintaining at least partially their stem cell character. In the case of inflammatory disease or trauma where the liberation and exposure to LPS will be increased, the expansion of NSPCs could be a first step towards regeneration of the ENS. The reduced and altered differentiation, as well as the induction of cytokine signalling, demonstrates that the stem cell niche may take part in the LPS-transmitted inflammatory processes in a direct and defined way.

Prolonged cultivation of hippocampal neural precursor cells shifts their differentiation potential and selects for aneuploid cells.

Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells.

Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve.

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Pharmacokinetics and ex vivo pharmacodynamic antimalarial activity of dihydroartemisinin-piperaquine in patients with uncomplicated falciparum malaria in Vietnam.

Compared to healthy subjects, malaria patients show a reduction in the mean oral clearance (1.19 versus 5.87 liters/h/kg of body weight) and apparent volume of distribution (1.47 versus 8.02 liters/kg) of dihydroartemisinin in Vietnamese patients following treatment with dihydroartemisinin-piperaquine (Artekin) for uncomplicated Plasmodium falciparum. Dihydroartemisinin is responsible for most of the ex vivo antimalarial activity of dihydroartemisinin-piperaquine.