Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.     

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly?s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.     

Antenna ring around trimeric Photosystem I in chlorophyll b containing cyanobacterium Prochlorothrix hollandica

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called “prochlorophytes”) that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.     

On the polyphasic quenching kinetics of chlorophyll a fluorescence in algae after light pulses of variable length

This study reports on kinetics of the fluorescence decay in a suspension of the alga Scenedesmus quadricauda after actinic illumination. These are monitored as the variable fluorescence signal in the dark following light pulses of variable intensity and duration. The decay reflects the restoration of chlorophyll fluorescence quenching of the photosystem II (PSII) antennas and shows a polyphasic pattern which suggests the involvement of different processes. The overall quenching curve after a fluorescence-saturating pulse (SP) of 250-ms duration, commonly used in pulse amplitude modulation applications as the tool for estimating the maximal fluorescence (F _m), has been termed P–O, in which P and O have the same meaning as used in the OJIP induction curve in the light. Deconvolution of this signal shows at least three distinguishable exponential phases with reciprocal rate constants of the order of 10, 10², and 10³ ms. The size of the long (>10³ ms) and moderate (~10² ms) lasting components relative to the complete quenching signal after an SP increases with the duration of the actinic pulse concomitantly with an increase in the reciprocal rate constants of the fast (~10 ms) and moderate quenching phases. Fluorescence responses upon single turnover flashes of 30-μs duration (STFs) given at discrete times during the P–O quenching were used as tools for identifying the quencher involved in the P–O quenching phase preceding the STF excitation. Results are difficult to interpret in terms of a single-hit two-state trapping mechanism with distinguishable quenching properties of open and closed reaction centers only. They give support for an earlier hypothesis on a double-hit three-state trapping mechanism in which the so-called semi-closed reaction centers of PSII are considered. In these trapping-competent centers the single reduced acceptor pair [PheQ _A]^1?, depending on the size of photoelectrochemically induced pH effects on the Q _B-binding site, functions as an efficient fluorescence quencher.     

Diagnostic Tests for Primary Renal Hypouricemia

Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 μmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.     

Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase

Six AChE monooxime-monocarbamoyl reactivators with an (E)-but-2-ene linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and insecticide paraoxon was tested in vitro. The reactivation efficacies of pralidoxime, HI-6, obidoxime, K048, K075 and the newly prepared reactivators were compared. According to the results obtained, one reactivator seems to be promising against tabun-inhibited AChE and two reactivators against paraoxon-inhibited AChE. The best results were obtained for bisquaternary substances with at least one oxime group in position four.     

Prdm9 deficiency of rat oocytes causes synapsis among non-homologous chromosomes and aneuploidy

Mammalian Genome - Aneuploidy (abnormal chromosome number) accompanies reduced ovarian function in humans and mice, but the reasons behind this concomitance remain underexplored. Some variants in...     

GRACE Score among Six Risk Scoring Systems (CADILLAC,PAMI, TIMI,Dynamic TIMI,Zwolle) Demonstrated the Best Predictive Value for Prediction of Long-Term Mortality in Patients with ST-Elevation Myocardial Infarction

Aim

To compare the prognostic accuracy of six scoring models for up to three-year mortality and rates of hospitalisation due to acute decompensated heart failure (ADHF) in STEMI patients.

Methods and Results

A total of 593 patients treated with primary PCI were evaluated. Prospective follow-up of patients was ≥3 years. Thirty-day, one-year, two-year, and three-year mortality rates were 4.0%, 7.3%, 8.9%, and 10.6%, respectively. Six risk scores—the TIMI score and derived dynamic TIMI, CADILLAC, PAMI, Zwolle, and GRACE—showed a high predictive accuracy for six- and 12-month mortality with area under the receiver operating characteristic curve (AUC) values of 0.73–0.85. The best predictive values for long-term mortality were obtained by GRACE. The next best-performing scores were CADILLAC, Zwolle, and Dynamic TIMI. All risk scores had a lower prediction accuracy for repeat hospitalisation due to ADHF, except Zwolle with the discriminatory capacity for hospitalisation up to two years (AUC, 0.80–0.83).

Conclusions

All tested models showed a high predictive value for the estimation of one-year mortality, but GRACE appears to be the most suitable for the prediction for a longer follow-up period. The tested models exhibited an ability to predict the risk of ADHF, especially the Zwolle model.