Development of an Automated MALDI Mass Spectrometry Assay for direct Analysis of Cellular Drug Uptake via the Organic Anion Transporting Polypeptide OATP2B1

For the longest time, there was the widely held belief that drug uptake into a cell is mainly due to diffusion, channels and carriers. Only in the 1940s, there was the first drug-transporter interaction discovered. As more transporter-related diseases where discovered and transport proteins identified that had an important connection to cancer, like the breast cancer resistance protein, the research field gained more interest. Drug-Drug interactions were identified and an International Transporter Consortium was founded that had the task to identify relevant transport proteins and closely work together with the drug approval by the FDA. The majority of transport proteins either belong to the class of ABC transporters, which are primary active export transporters or to the class of solute carrier (SLC) transporters, which are secondary active uptake transporters. OATP2B1 is an organic anion transporting polypeptide and is part of the group of SLC transporters. It is seen as an emerging transport protein and has gained attention due to many drug-fruit juice interactions. As the simultaneous intake of fruit juices and drugs are likely to happen in everyday life and OATP2B1 is a human transporter expressed mostly in liver and intestine, it is important to understand more about its uptake mechanism and possible inhibition. Until now, the emerging field of transporters are examined by either radioactive or fluorescence-based assays. Radioactive assays render a rather unpopular method with many obstacles like cost, safety-issues, labelling and no possibility of high-throughput. Fluorescence-based assays are widespread and have the positive property that they can be automated and used in HTS. The negative aspects here are also labelling and the false negatives and positives prediction that comes through autofluorescence and quenching effects. Our goal therefore was to develop an alternative cell based assay based on a different technology: MALDI MS. MALDI MS brings the advantage that it is a label-free technique and it also is HTS-compatible, like it was already shown in the past. Cell-based MALDI MS assays have gained recognition in the last years in consequence of their speed, robustness and ease in setup and are suitable for the investigation of transport mechanisms due to the abundance of additional information gathered by this technique. For the MALDI MS method development, the use of E3S as a substrate provided the best results. The optimal matrix composition for the detection of E3S in the cells had to be found. 2.5 mg/mL Ph-CCA-NH2 in 70 % ACN were identified as the best composition and were further used for transport characterisation with the confirmation of time-dependence and concentration-dependence of E3S uptake. The optimal assay conditions (2 min, 10 µM E3S) were used to screen a set of 294 compounds consisting of the top 300 marketed drugs and a set of compounds that are known to interact with OATP2B1. The used compounds were tested in their ability to inhibit the uptake of E3S into the cells. There were 76 compounds found with an inhibition of more than 50 %, which were then further analysed by pIC50 determination. 67 of those compounds could be verified as hits, leading also to 14 very potent inhibitors with a pIC50 over 6. With an average CV % under 10 for 6 biological replicates, the method confirms reproducibility and reliability of the data. As a reference assay to the aspired MALDI MS assay, also a fluorescence-based assay was developed to examine the uptake of DBF through OATP2B1. This assay was also used to screen the 294 compound set and 67 compounds with an inhibition ≥50 % were identified in the course of the experiments. 57 could be verified as a hit. There was a calculation being done to examine the clinical relevance of those transporters showing a clinical relevance of more than 60 % in the intestine reinforcing the meaning of transporter studies. By the comparison of the two techniques, it was found that only 47 inhibitors overlapped leading to compounds that were not found with one of both methods. This can eventually be explained by the use of different substrates and the multiple binding sites of OATP2B1, but still has to be addressed further. The comparison of the data with the previously published Karlgren et al. data set showed an overlap of 83 % and therefore shows the applicability of the MALDI MS method. To conclude, there were two assay systems developed that are suitable to examine the emerging transport protein OATP2B1. The importance of this transport protein has been shown through the amount of identified (clinically relevant) inhibitors. While the developed fluorescence-based method acts as a good reference method, the newly developed MALDI MS method represents a completely new way to analyse substrate and inhibitor of transporters. With its ease and speed in handling and most notably the label-free approach, the MALDI MS method is an indispensable tool for transporter characterisation and DDI analysis

The miR-223 host non-coding transcript linc-223 induces IRF4 expression in acute myeloid leukemia by acting as a competing endogenous RNA

Alterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long non-coding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic differentiation. Moreover, linc-223 expression inhibits cell cycle progression and promotes monocytic differentiation of AML cells. We also demonstrate that endogenous linc-223 localizes in the cytoplasm and acts as a competing endogenous RNA for miR-125-5p, an oncogenic microRNA in leukemia. In particular, we show that linc-223 directly binds to miR-125-5p and that its knockdown increases the repressing activity of miR-125-5p resulting in the downregulation of its target interferon regulatory factor 4 (IRF4), which it was previously shown to inhibit the oncogenic activity of miR-125-5p in vivo. Furthermore, data from primary AML samples show significant downregulation of linc-223 in different AML subtypes. Therein, these findings indicate that the newly identified lncRNA linc-223 may have an important role in myeloid differentiation and leukemogenesis, at least in part, by cross-talking with IRF4 mRNA

METTL3 regulates WTAP protein homeostasis

The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex

Octopamine and dopamine mediate waggle dance following and information use in honeybees

Honeybees can be directed to profitable food sources by following waggle dances performed by other bees. Followers can often choose between using this social information or relying on memories about food sources they have visited in the past, so-called private information. While the circumstances that favour the use of either social or private information have received considerable attention, still little is known about the neurophysiological basis of information use. We hypothesized that octopamine and dopamine, two biogenic amines with important functions in reward signalling and learning, affect dance use in honeybees. We orally administered octopamine and dopamine when bees collected food at artificial feeders and tested if this affected interest in dance information about a new food source. We predicted that octopamine reduces interest in dances and strengthens private information use via an increase in the perceived value of the previously exploited resource. Since dopamine has been shown to lower reward perception, we expected it to act in the opposite direction. Octopamine-treated foragers indeed followed 32% fewer dances than control bees and increased the use of private information. Conversely, dopamine-treated bees followed dances 15% longer than control bees, but surprisingly did not use social information more. Overall, our results suggest that biogenic amine signalling affects interactions among dancers and dance followers and, thus, information flow about high-quality food sources

Crossing the Divide: A Phenomenological Study of Early Childhood Literacy Teachers Who Choose To Work With Children In High Poverty Schools

In this phenomenological study, I explore the lived experiences of five early childhood educators, teaching literacy in high poverty schools. My work is guided by the research question: "What are the lived experiences of White early literacy teachers who choose to work with minority children in high poverty schools?" As phenomenology demands, my work is grounded in philosophy, and I turn to the writings of Sartre, Levinas, Derrida, Levin, and Gadamer. For methodological guidance, I rely on the work of Max van Manen. Through the voices of my participants, I excavate the meaning beneath their experiences. In my initial conversations with two of my participants, Will and Paula, I detect their chosen dedication to working in Title I schools, and their respect for children's individual needs and multiple identities. Identity continues to emerge as a central structure, both in the lived experiences of each of my five participants and in their pedagogical practice. While each of my participants is White, each one conveys a sense of having a multiplicity of identities, which enables them to connect with their students and families. Through their pedagogy, my participants also attend to the various aspects of their students' identities. They address needs relating to language, family, literacy, community, and the difficult choices and challenges that await their students in society. Throughout our conversations, the notions of choice and of crossing boundaries remain central. These teachers choose to work in low-income communities, where they move back-and-forth from their middle-class homes. As they teach a transformative curriculum, they also cross the boundary of curriculum, as they attend to the demands of standardization. Finally, I suggest that teacher education programs examine how boundary-crossing might inform their own pedagogical practice. Through pre-service teachers' exploration of their own identities and their experiences with families in Title I neighborhoods, they can become boundary-crossers, comfortably moving between two divided worlds. And, by learning how to integrate reading, writing, listening, and speaking throughout all aspects of the curriculum, they can teach children about the socially transformative power of literacy

4-(3-Nitrophenyl)thiazol-2-ylhydrazone derivatives as antioxidants and selective hMAO-B inhibitors: synthesis, biological activity and computational analysis

A new series of 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives were designed, synthesised, and evaluated to assess their inhibitory effect on the human monoamine oxidase (hMAO) A and B isoforms. Different (un)substituted (hetero)aromatic substituents were linked to N1 of the hydrazone in order to establish robust structure–activity relationships. The results of the biological testing demonstrated that the presence of the hydrazothiazole nucleus bearing at C4 a phenyl ring functionalised at the meta position with a nitro group represents an important pharmacophoric feature to obtain selective and reversible human MAO-B inhibition for the treatment of neurodegenerative disorders. In addition, the most potent and selective MAO-B inhibitors were evaluated in silico as potential cholinesterase (AChE/BuChE) inhibitors and in vitro for antioxidant activities. The results obtained from molecular modelling studies provided insight into the multiple interactions and structural requirements for the reported MAO inhibitory properties

Travelers' vaccinations: experience from the Travelers' Clinic of Hospital das Clínicas, University of São Paulo School of Medicine

O perfil dos indivíduos, a situação vacinal e as vacinas recomendadas aos viajantes que procuram o serviço médico de orientação pré-viagem do Ambulatório dos Viajantes do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo foram analisados no presente estudo. Dos 445 viajantes estudados, 51% eram mulheres; a mediana de idade foi de 33,5 anos; 51% viajavam a trabalho e 39,5% por lazer. Destinos mais procurados: África (47%); Ásia (31,7%); América do Sul (21,4%). Trezentos e oitenta e cinco (86,5%) viajantes tiveram indicação de vacinação para viagem. Principais vacinas recomendadas: febre tifóide (55,7%), difteria-tétano (54,1%), hepatite A (46,1%), hepatite B (44,2%), febre amarela (24,7%). A orientação pré-viagem mostrou-se importante não só para indicar as vacinas recomendadas para a viagem, mas também como oportunidade para atualização das vacinas de rotina.TThe profile and vaccination status of travelers seeking pre-travel medical advice at the Travelers' Clinic of Hospital das Clínicas, University of São Paulo School of Medicine, and the vaccines recommended for them, were analyzed in the present study. Among the 445 travelers who were studied, 51% were women, the median age was 33.5 years, 51% were traveling on business and 39.5% were traveling for leisure purposes. The destinations most sought were Africa (47%), Asia (31.7%) and South America (21.4%). Vaccination before traveling was recommended for 385 (86.5%) of the travelers. The main vaccines recommended were against typhoid fever (55.7%), diphtheria-tetanus (54.1%), hepatitis A (46.1%), hepatitis B (44.2%) and yellow fever (24.7%). The pre-travel guidance was shown to be important not only for indicating the vaccines recommended for the trip, but also as an opportunity to update routine vaccinations

About time:Ageing influences neural markers of temporal predictability

Timing abilities help organizing the temporal structure of events but are known to change systematically with age. Yet, how the neuronal signature of temporal predictability changes across the age span remains unclear. Younger (n = 21; 23.1 years) and older adults (n = 21; 68.5 years) performed an auditory oddball task, consisting of isochronous and random sound sequences. Results confirm an altered P50 response in the older compared to younger participants. P50 amplitudes differed between the isochronous and random temporal structures in younger, and for P200 in the older group. These results suggest less efficient sensory gating in older adults in both isochronous and random auditory sequences. N100 amplitudes were more negative for deviant tones. P300 amplitudes were parietally enhanced in younger, but not in older adults. In younger participants, the P50 results confirm that this component marks temporal predictability, indicating sensitive gating of temporally regular sound sequences.</p