1 July 2007 , Number 2

Iran has energy that China needs and Beijing provides a counter balance to western pressures on Tehran. The benefits are clear, but so are the risks for a rising power in the labyrinthine politics of the Middle East.

6 May 2020

Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic myeloid progenitors. Aberrantly activated tyrosine kinases (TK) are involved in leukemogenesis and are associated with poor treatment outcome. Kinase inhibitor (KI) treatment has shown promise in improving patient outcome in AML. However, inhibitor selection for patients is suboptimal.

In a preclinical effort to address KI selection, we analyzed a panel of 16 AML cell lines using phosphotyrosine (pY) enrichment-based, label-free phosphoproteomics. The Integrative Inferred Kinase Activity (INKA) algorithm was used to identify hyperphosphorylated, active kinases as candidates for KI treatment, and efficacy of selected KIs was tested.

Heterogeneous signaling was observed with between 241 and 2764 phosphopeptides detected per cell line. Of 4853 identified phosphopeptides with 4229 phosphosites, 4459 phosphopeptides (4430 pY) were linked to 3605 class I sites (3525 pY). INKA analysis in single cell lines successfully pinpointed driver kinases (PDGFRA, JAK2, KIT and FLT3) corresponding with activating mutations present in these cell lines. Furthermore, potential receptor tyrosine kinase (RTK) drivers, undetected by standard molecular analyses, were identified in four cell lines (FGFR1 in KG-1 and KG-1a, PDGFRA in Kasumi-3, and FLT3 in MM6). These cell lines proved highly sensitive to specific KIs. Six AML cell lines without a clear RTK driver showed evidence of MAPK1/3 activation, indicative of the presence of activating upstream RAS mutations. Importantly, FLT3 phosphorylation was demonstrated in two clinical AML samples with a FLT3 internal tandem duplication (ITD) mutation.

Our data show the potential of pY-phosphoproteomics and INKA analysis to provide insight in AML TK signaling and identify hyperactive kinases as potential targets for treatment in AML cell lines. These results warrant future investigation of clinical samples to further our understanding of TK phosphorylation in relation to clinical response in the individual patient.

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g. in general <100x) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g. 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2500 proteins and precise quantification of ~1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.

Signaling networks process intra- and extracellular information to modulate the functions of a cell. Deregulation of signaling networks results in abnormal cellular physiological states and often drives diseases. Network responses to a stimulus or a drug treatment can be highly heterogeneous across cells in a tissue because of many sources of cellular genetic and non-genetic variance. Signaling network heterogeneity is the key to many biological processes, such as cell differentiation and drug resistance. Only recently, the emergence of multiplexed single-cell measurement technologies has made it possible to evaluate this heterogeneity. In this review, we categorize currently established single-cell signaling network profiling approaches by their methodology, coverage, and application, and we discuss the advantages and limitations of each type of technology. We also describe the available computational tools for network characterization using single-cell data and discuss potential confounding factors that need to be considered in single-cell signaling network analyses.

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18–labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products.

Invitation Only Research Event

Invitation Only Research Event

Event participants

Dr Tedros Adhanom Ghebreyesus, Director-General, World Health Organization 

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.

tRNAs universally carry a CCA nucleotide triplet at their 3'-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

A. A. Vladimirov
Trans. Moscow Math. Soc. 80 (2020), 211-219.
Abstract, references and article information

Aos primeiros sinais de visão embaçada, as hipóteses mais frequentes sempre são miopia, astigmatismo, hipermetropia. Mas esses sintomas podem indicar outra doença ocular chamada ceratocone - uma deformidade progressiva da córnea, que assume o formato...

The post Visão embaçada e distorcida nem sempre é miopia: fique atento aos sinais do ceratocone appeared first on Saúde Próspera.

O que é Candidíase? Candidíase, é uma infecção sistêmica causada pelo fungos da Candida albicans. A Candida albicans é um tipo de fungo (levedura) que vive em harmonia no organismo,…

The post Candidíase – Conheça as causas, sintomas e tratamentos appeared first on Saúde Próspera.

A person needing a kidney transplant may have a friend or relative who volunteers to be a living donor, but whose kidney is incompatible, forcing the person to wait for a transplant from a deceased donor. In the U.S. alone, thousands of people die each year without ever finding a suitable kidney. A new technique applies graph theory to groups of incompatible patient-donor pairs to create the largest possible number of paired-donation exchanges. These exchanges, in which a donor paired with Patient A gives a kidney to Patient B while a donor paired with Patient B gives to Patient A, will dramatically increase transplants from living donors. Since transplantation is less expensive than dialysis, this mathematical algorithm, in addition to saving lives, will also save hundreds of millions of dollars annually. Naturally there can be more transplants if matches along longer patient-donor cycles are considered (e.g., A.s donor to B, B.s donor to C, and C.s donor to A). The problem is that the possible number of longer cycles grows so fast hundreds of millions of A >B>C>A matches in just 5000 donor-patient pairs that to search through all the possibilities is impossible. An ingenious use of random walks and integer programming now makes searching through all three-way matches feasible, even in a database large enough to include all incompatible patient-donor pairs. For More Information: Matchmaking for Kidneys, Dana Mackenzie, SIAM News, December 2008. Image of suboptimal two-way matching (in purple) and an optimal matching (in green), courtesy of Sommer Gentry.

Mathematics contributes in many ways to the process of converting wind power into usable energy. Large-scale weather models are used to find suitable locations for wind farms, while more narrowly focused models incorporating interactions arising from factors such as wake effects and turbulence specify how to situate individual turbines within a farm. In addition, computational fluid dynamics describes air flow and drag around turbines. This helps determine the optimal shapes for the blades, both structurally and aerodynamically, to extract as much energy as possible, and keep noise levels and costs down. Mathematics also helps answer two fundamental questions about wind turbines. First, why three blades? Turbines with fewer blades extract less energy and are noisier (because the blades must turn faster). Those with more than three blades would capture more energy but only about three percent more, which doesn.t justify the increased cost. Second, what percentage of wind energy can a turbine extract? Calculus and laws of conservation provide the justification for Betz Law, which states that no wind turbine can capture more than 60% of the energy in the wind. Modern turbines generally gather 40-50%. So the answer to someone who touts a turbine that can capture 65% of wind energy is "All Betz" are off. For More Information: Wind Energy Explained: Theory, Design and Application, Manwell, McGowan, and Rogers, 2010.

There.s more mathematics involved in juggling than just trying to make sure that the number of balls (or chainsaws) that hits the ground stays at zero. Subjects such as combinatorics and abstract algebra help jugglers answer important questions, such as whether a particular juggling pattern can actually be juggled. For example, can balls be juggled so that the time period that each ball stays aloft alternates between five counts and one? The answer is Yes. Math also tells you that the number of balls needed for such a juggling pattern is the average of the counts, in this case three. Once a pattern is shown to be juggleable and the number of balls needed is known, equations of motion determine the speed with which each ball must be thrown and the maximum height it will attain. Obviously the harder a juggler throws, the faster and higher an object will go. Unfortunately hang time increases proportionally to the square root of the height, so the difficulty of keeping many objects in the air increases very quickly. Both math and juggling have been around for millennia yet questions still remain in both subjects. As two juggling mathematicians wrote, .A juggler, like a mathematician, is never finished: there is always another great unsolved problem.

There.s more mathematics involved in juggling than just trying to make sure that the number of balls (or chainsaws) that hits the ground stays at zero. Subjects such as combinatorics and abstract algebra help jugglers answer important questions, such as whether a particular juggling pattern can actually be juggled. For example, can balls be juggled so that the time period that each ball stays aloft alternates between five counts and one? The answer is Yes. Math also tells you that the number of balls needed for such a juggling pattern is the average of the counts, in this case three. Once a pattern is shown to be juggleable and the number of balls needed is known, equations of motion determine the speed with which each ball must be thrown and the maximum height it will attain. Obviously the harder a juggler throws, the faster and higher an object will go. Unfortunately hang time increases proportionally to the square root of the height, so the difficulty of keeping many objects in the air increases very quickly. Both math and juggling have been around for millennia yet questions still remain in both subjects. As two juggling mathematicians wrote, .A juggler, like a mathematician, is never finished: there is always another great unsolved problem.

In the wake of the COVID-19 crisis, any economic stimulus measures must safeguard nature or governments risk exposing humanity to further pandemics.

Digital technology was not invented to tackle inequality, and there is even a risk that it could widen existing economic and social disparities. But, as the case of China illustrates, new platforms also offer many possible ways to narrow the opportunity gap.

Esther McVey said the government will release local data on properties and land to help the proptech sector thrive

David Murray-Hundley, the Grumpy Entrepreneur, shares his six pieces of advice for founders of fast growing companies to help maintain control