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Dysregulation of the transcription factor MYC is involved in many human cancers. The dimeric transcription factor complexes of MYC/MAX and MAX/MAX activate or inhibit, respectively, gene transcription upon binding to the same enhancer box DNA. Targeting these complexes in cancer is a long-standing challenge. Inspired by the inhibitory activity of the MAX/MAX dimer, we engineered covalently linked, synthetic homo- and heterodimeric protein complexes to attenuate oncogenic MYC-driven transcription. We prepared the covalent protein complexes (∼20 kDa, 167–231 residues) in a single shot via parallel automated flow synthesis in hours. The stabilized covalent dimers display DNA binding activity, are intrinsically cell-penetrant, and inhibit cancer cell proliferation in different cell lines. RNA sequencing and gene set enrichment analysis in A549 cancer cells confirmed that the synthetic dimers interfere with MYC-driven transcription. Our results demonstrate the potential of automated flow technology to rapidly deliver engineered synthetic protein complex mimetics that can serve as a starting point in developing inhibitors of MYC-driven cancer cell growth., Via parallel automated flow synthesis, we prepared covalent dimeric protein analogues of the transcription factor MYC. The dimers can enter cells and inhibit MYC-driven cancer cell proliferation.

The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. To test this idea, we focused our study on nuclear condensates reported in cell lines, demonstrated they occur in normal human and malignant breast cancer, and developed assays to test clinically active antineoplastic small molecule drugs relative to these condensates.To study the behavior of drugs within condensates, these were modeled in vitro with purified proteins and visualized by fluorescent confocal microscopy. We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. For each drug, the small molecule partitioned into the same condensate in vitro in which its established target resides in vivo. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration.Alkylating agents are a class of commonly used antineoplastic compounds, of which cisplatin is a prominent example. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Thus, we conclude that cisplatin preferentially modifies transcriptional condensate-associated DNA in cells, and that this causes selective condensate disruption. The mechanisms that produce drug resistance can provide clues to drug activity in the clinical setting. Investigating the behavior of tamoxifen within ER transcriptional condensates demonstrated that it disrupts these condensates in vitro and on oncogenes in cells; hormonal therapy resistant ESR1 mutations render these condensates resistant. MED1 overexpression, a poorly understood mechanism of tamoxifen resistance, increased the size of ER-MED1 condensates, thereby rending tamoxifen more dilute and ineffective when concentrated therein. This suggest that altering the size and nature of transcription condensates in breast cancer can mediate drug resistance in the clinical setting.Our results show that antineoplastic drugs partition selectively into condensates, that this can occur through physicochemical properties independent of their molecular targets, and that resistance to drugs may occur through condensate altering mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment. Citation Format: Isaac Klein, Ann Boija, Lena Afeyan, Susana Wilson Hawken, Mengyang Fan, Alessandra Dall'Agnese, Ozgur Oksuz, Jonathan Henninger, Krishna Shrinivas, Benjamin Sabari, Ido Sagi, Victoria Clark, Jesse Platt, Mrityunjoy Kar, Patrick McCall, Alicia Zamudio, John Mantiega, Eliot Coffey, Charles Li, Nancy Hannett, Yang Guo, Tim-Michael Decker, Tong Lee, Tinghu Zhang, Jing-Ke Weng, Dylan Taatjes, Arup Chakraborty, Phillip Sharp, Young Tae Chang, Anthony Hyman, Nathanael Gray, Richard Young. Partitioning of cancer therapeutics in nuclear condensates [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-10.

Background: In analytical performance studies, the choice of comparator method plays an important role, as studies have shown that there exist relevant systematic differences (bias) between laboratory analyzers. The feasibility of retrospective recalibration of measurement results through comparison with methods or materials of higher metrological order to minimize bias was therefore assessed. Method: Existing data from performance studies of continuous and blood glucose monitoring systems were retrospectively analyzed. Comparison with a higher-order method was performed for two different data sets. In both cases, subject samples were measured, and a subset was also measured on a higher-order method. Recalibration based on higher-order materials (standard reference material [SRM]) was conducted for two different data sets containing results from SRM and subject samples. Linear regression analysis was performed for each device separately. Resulting equations were applied to the respective complete data set of subject samples. Bias between devices in a data set across all subject samples was assessed before and after recalibration. Results: Bias between devices was reduced from −3.6% to +0.6% in one data set and from +11.0% to +0.3% in the other by recalibration based on higher-order method. Using higher-order materials, bias was also reduced by recalibration, but mixed results were found: Bias was reduced from −3.1% to −0.1% in one data set and from −4.3% to −2.7% in the other. Conclusions: Recalibration did lead to a decrease in bias and thus can reduce the impact of the choice of comparator method. The procedure should be verified in a prospectively designed setting.

Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb-group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here, we show that the p300/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 μM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex1–4. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7–12. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9–12. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.

Background Creatinine measurement for estimation of glomerular filtration rate (GFR) is a frequently used laboratory test. Differences in analytic creatinine methods have caused large inter-laboratory variation. International and national standardization efforts have been made in the last decade. Methods This study describes the results of the standardization efforts in Sweden by summarizing data for creatinine concentration in blood plasma in the Equalis quality assessment program during 1996–2014. Results Non-compensated Jaffe methods dominated in 1996–2001 (91 of 103 laboratories; 90%) and were then gradually replaced by either compensated Jaffe methods or enzymatic creatinine methods. In 2014 a majority of Swedish hospital laboratories (139 of 159; 87%) used enzymatic methods. The reported mean creatinine value by the Swedish laboratories was about 10 μmol/L higher than the isotope dilution mass spectrometry (IDMS) assured reference value in 2003, but consistent with the reference value from 2009 to 2014. The inter-laboratory CV was 7%–9% for creatinine values until 2007, and thereafter gradually decreased to about 4%–5% in 2014. Conclusions The introduction of enzymatic methods in Swedish laboratories has contributed to achieving a low inter-laboratory variation. Also, the reported values are lower for enzymatic methods compared to Jaffe methods, and the values obtained with enzymatic methods were consistent with IDMS certified values established at reference laboratories. Thus, many Swedish hospital laboratories reported 10 μmol/L lower, and more true, creatinine concentrations in 2012 than in 2003, which may cause bias in longitudinal studies.

Det har blivit allt mer viktigt för företag att ta ansvar inom områdena miljö, ekonomi och samhälle. Företag använder sig av CSR-kommunikation för att visa vilka handlingar de vidtar för att uppfylla dessa förväntningar och erhålla legitimitet. Det förekommer dock att CSR-kommunikationen inte överensstämmer med verkligheten. Det skapas då ett gap mellan företagens symboliska och substantiva handlingar, vilket benämns som CSR-decoupling. Företag i USA kontra Västeuropa befinner sig på olika marknader när det kommer till företagskulturer. Denna studie syftar därför till att undersöka användandet av CSR-decoupling i USA och Västeuropa. Studien undersöker även verkställande direktörens makt och intressenternas krav för att se om dessa faktorer påverkar användandet av CSR-decoupling. Studien är en kvantitativ tvärsnittsstudie som bygger på data bestående av amerikanska börsnoterade företag och västeuropeiska börsnoterade företag. Hypoteserna besvaras utifrån tidigare forskning och resultatet från en regressionsanalys samt t-test. Resultatet från regressionsanalysen visar att användandet av CSR-decoupling är större i USA än i Västeuropa, medan t-testet visar det motsatta. Av studien kunde inget samband fastställas mellan verkställande direktörens makt och CSR-decoupling. Resultatet visade däremot ett positivt samband mellan intressenternas krav och CSR-decoupling, samt att västeuropeiska börsnoterade företag påverkas mer av intressenternas krav när det kommer till användandet av decouplingstrategier. It has become increasingly important for companies to take responsibility in the areas of environment, economy and society. Companies use CSR-communication to show what actions they are taking to meet these expectations and gain legitimacy. However, it happens that CSR-communication does not correspond to reality. This creates a gap between the companies' symbolic and substantive actions, which is referred to as CSR-decoupling. Companies in the U.S versus Western Europe are in different markets when it comes to corporate cultures. This study therefore aims to research the use of CSR-decoupling in the United States and Western Europe. We also examine the CEO's power and stakeholder requirements to see if these factors affect the use of CSR-decoupling. The study is a quantitative cross-sectional study based on data consisting of U.S listed companies and Western European listed companies. The hypotheses are answered based on previous research and the results of a regression analysis and t-test. The results from the regression analysis show that the use of CSR-decoupling is greater in the U.S than in Western Europe, while the t-test show the opposite. From the study, no statistical relationship could be established between the CEO's power and CSR-decoupling. The result, on the other hand, showed a positive statistical relationship between stakeholder requirements and CSR-decoupling, and that Western European listed companies are more affected by stakeholder requirements when it comes to the use of decoupling strategies.

In this thesis a regression analysis of ten independent data sets is analysed in order toestimate losses and Key Risk Indicators (KRI). Each data set contains a list of objects,impacts that each object contains and revenue stream values (RSV) to each impact.The project investigates the data and simulate yearly losses as response variables in theregression modelling. The three regressors that influence the yearly losses are numberof objects, sum of revenue streams and expected aggregated losses. Given the responsevariable from each data set a percentage scale of KRI’s is determined indicating howlarge losses each set possess. I denna avhandling analyseras en regressionsmodellering av tio oberoende mängderdata för att uppskatta förluster och Key Risk Indicators. Den givna dataupsättningeninnehåller en lista med objekt, påverkan varje objekt erhåller och vad respektiveobjekt omsätter. Projektet undersöker den givma datan och simulerar årliga förlustersom svarsvariabler i regressionsmodelleringen. De tre regressorerna som påverkarde årliga förlusterna är antalet objekt, summan av intäckterna och förvämtadesammanlagda förlusterna. Från den givna svarsvariabeln från varje datamängdbestäms en procentuell skala av KRIer som indikerar hur stora förluster varjeuppsättning har.

Malignant transformation is characterized by dysregulation of diverse cellular processes that have been the subject of detailed genetic, biochemical, and structural studies, but only recently has evidence emerged that many of these processes occur in the context of biomolecular condensates. Condensates are membrane-less bodies, often formed by liquid-liquid phase separation, that compartmentalize protein and RNA molecules with related functions. New insights from condensate studies portend a profound transformation in our understanding of cellular dysregulation in cancer. Here we summarize key features of biomolecular condensates, note where they have been implicated—or will likely be implicated—in oncogenesis, describe evidence that the pharmacodynamics of cancer therapeutics can be greatly influenced by condensates, and discuss some of the questions that must be addressed to further advance our understanding and treatment of cancer., NIH (Grants GM123511, CA213333 and CA155258)

Sensitivity and specificity of rapid antigen detection tests (RADTs) for detection of group A hemolytic streptococcus (GAS) vary. The purpose is to present the first SKUP (Scandinavian evaluation of laboratory equipment for point of care testing) evaluations concerning the assessment of the diagnostic performance and user-friendliness of two RADTs for detection of GAS when used under real-life conditions in primary health care. Throat samples were collected in duplicates at primary health care centers (PHCCs) from patients with symptoms of pharyngitis. The performance of QuickVue Dipstick Strep A test (307 samples) and DIAQUICK Strep A Blue Dipstick (348 samples) was evaluated using culture results at a clinical microbiology laboratory as comparison. The user-friendliness was evaluated using a questionnaire. The diagnostic sensitivity was 92% (90% confidence interval (CI) 87–96%) and 72% (90% CI 65–79%), while the diagnostic specificity was 86% (90% CI 81–90%) and 98% (90% CI 96–99%) for QuickVue Dipstick Strep A test and DIAQUICK Strep A Blue Dipstick, respectively. Both RADTs obtained acceptable assessments for user-friendliness and fulfilled SKUP’s quality goal for user-friendliness. The diagnostic sensitivity for QuickVue Dipstick Strep A test and the diagnostic specificity for DIAQUICK Strep A Blue Dipstick in this objective and supplier-independent evaluation were higher compared with previous meta-analyses of RADTs. However, the diagnostic specificity for QuickVue Dipstick Strep A test and the diagnostic sensitivity for DIAQUICK Strep A Blue Dipstick were lower compared with previous meta-analyses of RADTs.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors

Drug partitioning in nuclear condensates There is increasing interest in the function of phase-separated biomolecular condensates in cells because of their distinct properties and expanding roles in important biological processes. Klein et al. considered the fate of small-molecule therapeutics in the context of nuclear condensates (see the Perspective by Viny and Levine). They show that certain antineoplastic drugs have physicochemical properties that cause them to concentrate preferentially in condensates, both in vitro and in cancer cells. This property influences drug activity, and protein mutations that alter condensate formation can lead to drug resistance. Optimizing condensate partitioning may be valuable in developing improved therapeutics. Science , this issue p. 1386 ; see also p. 1314