Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family.

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle

Gain Scheduling for the Orion Launch Abort Vehicle Controller

One of NASAs challenges for the Orion vehicle is the control system design for the Launch Abort Vehicle (LAV), which is required to abort safely at any time during the atmospheric ascent portion of ight. The focus of this paper is the gain design and scheduling process for a controller that covers the wide range of vehicle configurations and flight conditions experienced during the full envelope of potential abort trajectories from the pad to exo-atmospheric flight. Several factors are taken into account in the automation process for tuning the gains including the abort effectors, the environmental changes and the autopilot modes. Gain scheduling is accomplished using a linear quadratic regulator (LQR) approach for the decoupled, simplified linear model throughout the operational envelope in time, altitude and Mach number. The derived gains are then implemented into the full linear model for controller requirement validation. Finally, the gains are tested and evaluated in a non-linear simulation using the vehicles ight software to ensure performance requirements are met. An overview of the LAV controller design and a description of the linear plant models are presented. Examples of the most significant challenges with the automation of the gain tuning process are then discussed. In conclusion, the paper will consider the lessons learned through out the process, especially in regards to automation, and examine the usefulness of the gain scheduling tool and process developed as applicable to non-Orion vehicles

Growth of Escherichia coli in human milk and powdered infant formula under various treatments and feeding conditions in neonatal units

Milk supplied to neonates in neonatal units is kept at room temperature for some time, which could influence microbial growth. This study aims to evaluate the growth of Escherichia coli in HM and PIF under various treatments and conditions, as well as to determine the influence of different thawing methods on microbial growth in HM. The number of E. coli generations appearing over a 4 h period at 22 °C in HM (frozen; frozen and pasteurized; and frozen, pasteurized, and fortified) and in PIF (four brands) was determined. E. coli counts in HM inoculated and thawed using different methods were also compared. In frozen HM and in pasteurized and frozen HM, significant differences were found after 2.5 h and 1.5 h, respectively. In PIF, differences were found between 1.5 and 3 h. With regard to the thawing process, the lowest microorganism counts were obtained at 4 °C overnight; thus, it seems advisable to store milk at room temperature for a maximum of 1 h during administration in neonatal units. Thawing HM at 4 °C overnight should be the method of choice

Proposal of a water-quality index for high andean basins: application to the Chumbao river, Andahuaylas, Peru

Thewater fromthe high Andean rivers is peculiar due to its composition and the geomorphology of its sources, and naturally or anthropogenically contamination is not discarded along its course. This water is used for agriculture and human consumption, therefore knowing its quality is important. This research aimed to proposing and formulate a water-quality index for high Andean basins through the Delphimethod, and its application in the Chumbao River located in Andahuaylas-Peru. Forty-three water-quality parameters were evaluated through the Delphi method, and the water-quality index (WQIHA) was formulated with a weighted average of the weights of the selected parameters, it was compared with the WQI Dinius. For this purpose, ten sampling points were considered along the Chumbao River located between 4274 and 2572 m of altitude and theWQIHA was applied. In addition, field and laboratory analyses were carried out in 2018, 2019, and 2021, in dry and rainy seasons. Twenty parameters were grouped in the physicochemical sub-index (SIPC), heavy metals sub-index (SIHM), and organic matter sub-index (SIOM). Each group contributed with weights of 0.30, 0.30, and 0.40, respectively, for theWQIHA formulation. The SIPC and SIOM showed that the areas near the head of the basin presented excellent and good quality, while the urbanized areas were qualified as marginal to poor; SIHM reported good quality in all points and seasons. Regarding the WQIHA, the index shows good quality in the zones above 3184 m of altitude, contrasting with poor quality downstream, decreasing notably in both seasons, suggesting continuous degradation of the water body

Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced micro- RNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these prodifferentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.Comment: 45 pages 5 figure

Interlaboratory analytical validation of a Next-generation sequencing strategy for clonotypic assessment and minimal residual disease monitoring in multiple myeloma

[Context]: Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. [Objective]: To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. [Design]: The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. [Results]: Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. [Conclusions]: Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures. Multiple myeloma (MM) is a plasma-cell dyscrasia characterized by the accumulation of plasma cells in the bone marrow that produces an excess of clonal immunoglobulins (M-protein or monoclonal component).1 New treatment approaches have increased the number of patients achieving complete response (CR),2–5 progressively improving progression-free and overall survival rates in the last 10 years.6–11 Nonetheless, the presence of low levels of drug-resistant cells (known as minimal residual disease, MRD)12–14 that remain undetected by conventional serologic and morphologic methods explains frequent relapses with this disease, which is still considered an incurable illness.Minimal residual disease is currently considered one of the most informative prognostic parameters, since those patients with undetectable disease have shown prolonged survival rates as compared with MRD-positive patients,15–17 and this difference is still significant even when patients achieving only stringent complete response (sCR) are taken into account.18 The International Myeloma Working Group (IMWG) defined MRD positivity as the persistence of clonal malignant plasma cells assessed with a sensitivity of at least 10−5 (1 malignant cell per hundred thousand normal cells)19 ; therefore, MRD should be monitored with only highly sensitive methods. To date, 3 different approaches have been tested for MRD monitoring in hematologic malignancies: immunophenotypic (multiparametric flow cytometry [MFC]),20 molecular (quantitative polymerase chain reaction [PCR], next-generation sequencing [NGS], digital PCR),21–23 and imaging tools (positron emission tomography–computed tomography; magnetic resonance imaging).24,25 However, in MM standardization has been achieved only for MFC26 and NGS.27,28 As a result, the IMWG recommended the use of highly sensitive, standardized flow and sequencing approaches,19 including EuroFlow's next-generation flow (NGF)29 and Adaptive Biotechnologies' ClonoSEQ solutions (Adaptive Biotechnologies, Seattle, Washington). NGF is a 2-tube, 8-color flow assay that allows the simultaneous analysis of 10 million cells, providing a sensitivity of around 2·10−6.This work was partially supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01956, CIBERONC-CB16/12/00233, and “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010). García-Álvarez, Prieto-Conde, and Jiménez were supported by the Fundación Española de Hematología y Hemoterapia (FEHH, cofunded by Fundación Cris in the latter case), Medina by the European Social Fund through the University of Salamanca and the ISCIII (FI19/00320), and Sarasquete by the ISCIII (CPII18/00028). All Spanish funding is cosponsored by the European Union FEDER program