Dysregulation of phosphorylation-mediated signaling drives the initiation and progression of many diseases. A substrate-specific kinase assay capable of quantifying the altered site-specific phosphorylation of its phenotype-dependent substrates provides better specificity to monitor a disease state. We report a sensitive and rapid substrate-specific kinase assay by integrating site-specific peptide reporter and multiple reaction monitoring (MRM)-MS platform for relative and absolute quantification of substrate-specific kinase activity at the sensitivity of nanomolar kinase and nanogram cell lysate. Using non-small cell lung cancer as a proof-of-concept, three substrate peptides selected from constitutive phosphorylation in tumors (HDGF-S165, RALY-S135, and NRD1-S94) were designed to demonstrate the feasibility. The assay showed good accuracy (<15% nominal deviation) and reproducibility (<15% CV). In PC9 cells, the measured activity for HDGF-S165 was 3.2 ± 0.2 fmol μg−1 min−1, while RALY-S135 and NRD1-S94 showed 4- and 20-fold higher activity at the sensitivity of 25 ng and 5 ng lysate, respectively, suggesting different endogenous kinases for each substrate peptide. Without the conventional shotgun phosphoproteomics workflow, the overall pipeline from cell lysate to MS data acquisition only takes 3 h. The multiplexed analysis revealed differences in the phenotype-dependent substrate phosphorylation profiles across six NSCLC cell lines and suggested a potential association of HDGF-S165 and NRD1-S94 with TKI resistance. With the ease of design, sensitivity, accuracy, and reproducibility, this approach may offer rapid and sensitive assays for targeted quantification of the multiplexed substrate-specific kinase activity of small amounts of sample.
Drug development is a long, expensive and multistage process geared to achieving safe drugs with high efficacy. A crucial prerequisite for completing the medication regimen for oral drugs, particularly for pediatric and geriatric populations, is achieving taste that does not hinder compliance. Currently, the aversive taste of drugs is tested in late stages of clinical trials. This can result in the need to reformulate, potentially resulting in the use of more animals for additional toxicity trials, increased financial costs and a delay in release to the market. Here we present BitterIntense, a machine learning tool that classifies molecules into “very bitter” or “not very bitter”, based on their chemical structure. The model, trained on chemically diverse compounds, has above 80% accuracy on several test sets. Our results suggest that about 25% of drugs are predicted to be very bitter, with even higher prevalence (~40%) in COVID19 drug candidates and in microbial natural products. Only ~10% of toxic molecules are predicted to be intensely bitter, and it is also suggested that intense bitterness does not correlate with hepatotoxicity of drugs. However, very bitter compounds may be more cardiotoxic than not very bitter compounds, possessing significantly lower QPlogHERG values. BitterIntense allows quick and easy prediction of strong bitterness of compounds of interest for food, pharma and biotechnology industries. We estimate that implementation of BitterIntense or similar tools early in drug discovery process may lead to reduction in delays, in animal use and in overall financial burden.
Bdellovibrio and like organisms (BALOs) prey on Gram-negative bacteria in the planktonic phase as well as in biofilms, with the ability to reduce prey populations by orders of magnitude. During the last few years, evidence has mounted for a significant ecological role for BALOs, with important implications for our understanding of microbial community dynamics as well as for applications against pathogens, including drug-resistant pathogens, in medicine, agriculture and aquaculture, and in industrial settings for various uses. However, our understanding of biofilm predation by BALOs is still very fragmentary, including gaps in their effect on biofilm structure, on prey resistance, and on evolutionary outcomes of both predators and prey. Furthermore, their impact on biofilms has been shown to reach beyond predation, as they are reported to reduce biofilm structures of non-prey cells (including Gram-positive bacteria). Here, we review the available literature on BALOs in biofilms, extending known aspects to potential mechanisms employed by the predators to grow in biofilms. Within that context, we discuss the potential ecological significance and potential future utilization of the predatory and enzymatic possibilities offered by BALOs in medical, agricultural and environmental applications.
International investment agreements (IIAs) are a significant manifestation of the impact of legal globalization on national public policy. These are thousands of (mostly) bilateral treaties through which states commit to protect the rights of foreign investors. Moreover, these obligations can be enforced by a system of binding international investor-state dispute settlement (ISDS), which allows investors to file claims against host countries that allegedly violated their obligations under their IIAs. The legitimacy of IIAs and ISDS is highly contested, however. On the one hand, they encroach on states’ regulatory space (SRS) and delegate legal authority to ad-hoc arbitration bodies, which lack transparency and accountability. On the other hand, their alleged positive effect on foreign investment is uncertain. As a party to about forty IIAs, Israel’s SRS is certainly affected by IIAs. Such potential impact came to the fore when an American company, Noble Energy, indicated that it might turn to ISDS against the Israeli government in relations to a disputed gas exploration project. This article examines the implications of IIAs and ISDS to SRS both globally and with respect to Israel. After elaborating on and illustrating these relationships in the global arena, we present a measure of SRS that facilitates a systematic comparison of IIAs across time and space on this key dimension. We show that, of late, states around the world conclude IIAs with greater regulatory space and that Israel tracks this global trend. A legal analysis of two investment disputes in the energy sector suggests that Israeli IIAs expose the country to costly ISDS claims and potentially limit its ability to regulate in important policy areas. We conclude that Israel will do well to sign or renegotiate IIAs with greater regulatory space.
Abstract With climate warming, drought becomes a vital challenge for agriculture. Extended drought periods affect plant?pathogen interactions. We demonstrate an interplay in tomato between drought and infection with tomato yellow leaf curl virus (TYLCV). Infected plants became more tolerant to drought, showing plant readiness to water scarcity by reducing metabolic activity in leaves and increasing it in roots. Reallocation of osmolytes, such as carbohydrates and amino acids, from shoots to roots suggested a role of roots in protecting infected tomatoes against drought. To avoid an acute response possibly lethal for the host organism, TYLCV down-regulated the drought-induced activation of stress response proteins and metabolites. Simultaneously, TYLCV promoted the stabilization of osmoprotectants' patterns and water balance parameters, resulting in the development of buffering conditions in infected plants subjected to prolonged stress. Drought-dependent decline of TYLCV amounts was correlated with HSFA1-controlled activation of autophagy, mostly in the roots. The tomato response to combined drought and TYLCV infection points to a mutual interaction between the plant host and its viral pathogen.
Reclaimed wastewater is increasingly used to irrigate agriculture crops. We have previously shown that carbamazepine (CBZ), an anticonvulsant human medication, not entirely discarded during wastewater purification, induces a stress response in tomatoes grown with roots bathing in CBZ-containing water. Induction of stress-related osmoprotectants (sugars, amino acids, proteins) was conspicuous in CBZ-treated seedlings. Here, tomato seedlings were grown in pots watered with increasing concentrations of CBZ. Soluble sugars effectively reacted to CBZ in both leaves and roots. However, the induction of stress-related amino acids and proteins was relevant in roots, but insignificant in leaves. Therefore, roots may be the site where CBZ stress is exerted. Moreover, roots may protect the whole plant from the pharmaceutical. Tomato crops endure biotic stresses, caused by whitefly (Bemisia tabaci) infestation and by the tomato yellow leaf curl begomovirus (TYLCV) they vector. The interplay between CBZ, TYLCV infection and B. tabaci was studied in tomato. Whiteflies preferred CBZ-treated plantlets than control plants, which may be due to increased amount of sugars in leaves. The increased amount of viruliferous whiteflies on CBZ-treated plants is expected to be accompanied by a rise in virus amounts. In fact, CBZ caused a reduction of TYLCV amounts. CBZ-dependent activation of autophagy degradation may explain this decrease in virus amounts. TYLCV infection mitigates the activation of stress markers associated with CBZ treatment. Altogether, CBZ in the water used to irrigate tomatoes grown in pots causes a relatively weak plant stress response, but is definitively sensed by insect and by virus.
Reclaimed wastewater is increasingly used to irrigate agriculture crops. We have previously shown that carbamazepine (CBZ), an anticonvulsant human medication, not entirely discarded during wastewater purification, induces a stress response in tomatoes grown with roots bathing in CBZ-containing water. Induction of stress-related osmoprotectants (sugars, amino acids, proteins) was conspicuous in CBZ-treated seedlings. Here, tomato seedlings were grown in pots watered with increasing concentrations of CBZ. Soluble sugars effectively reacted to CBZ in both leaves and roots. However, the induction of stress-related amino acids and proteins was relevant in roots, but insignificant in leaves. Therefore, roots may be the site where CBZ stress is exerted. Moreover, roots may protect the whole plant from the pharmaceutical. Tomato crops endure biotic stresses, caused by whitefly (Bemisia tabaci) infestation and by the tomato yellow leaf curl begomovirus (TYLCV) they vector. The interplay between CBZ, TYLCV infection and B. tabaci was studied in tomato. Whiteflies preferred CBZ-treated plantlets than control plants, which may be due to increased amount of sugars in leaves. The increased amount of viruliferous whiteflies on CBZ-treated plants is expected to be accompanied by a rise in virus amounts. In fact, CBZ caused a reduction of TYLCV amounts. CBZ-dependent activation of autophagy degradation may explain this decrease in virus amounts. TYLCV infection mitigates the activation of stress markers associated with CBZ treatment. Altogether, CBZ in the water used to irrigate tomatoes grown in pots causes a relatively weak plant stress response, but is definitively sensed by insect and by virus.
AIM: We aimed to explore how interpreters experienced being part of a special designed health introduction course and to obtain their perspectives on the learning process. The overall aim was to improve health care to patients with language barriers. DESIGN: With a hermeneutic phenomenological approach, we used participant observations as a method for collecting data. METHOD: The teaching methods used were case-based learning, role-play and active involvement including dialogs, discussions and critical reflections. The interpreters were divided into teams of 10-15 interpreters. Over the duration of 6 weeks, teaching sessions were conducted, with 3 hr/week planned. RESULTS: The findings emphasize the importance of providing the interpreters with education, supervision and a work environment where they can confidentially share and get competent feedback on their experiences and linguistic skills, in order to ensure patients get the correct information in their native language.
Microvilli generate the small intestinal brush border, the main site of nutrient digestion and absorption. Mucosal structuring of the small intestine of chicken during the perihatch period has been widely researched, yet the developmental dynamics of micro-villi during this period have not been fully character-ized. In this study, we examined the structural and molecular characteristics of microvilli assembly and maturation during the perihatch period. Small intes-tines of broiler embryos and chicks were sampled at prehatch ages 17 E and 19 E, at day of hatch (DOH) and at 1, 3, 7, and 10 d posthatch. Morphological evalu-ations and measurements were conducted by scanning electron microscopy (SEM) and light microscopy (LM) (n = 3/timepoint), and expression of microvilli structural genes Plastin 1, Ezrin, and Myo1a was exam-ined by Real-Time qPCR (n = 6/timepoint). Results revealed dissimilar patterns of microvilli and villi devel-opment during the perihatch period. From 19 E to 1 d, microvilli lengths increased 4.3-fold while villi lengths increased 2.8-fold (P < 0.0001). From 3 to 7 d, villi lengths increased by 20% (P < 0.005), while microvilli lengths decreased by 41% (P = 0.001). At 10 d, micro-villi lengths stabilized, while villi continued to elongate by 26% (P < 0.0001). Estimations of the microvilli amplification factor (MAF) and total enterocyte surface area (TESA) revealed similar trends, with peak values of 78.53 and 1961.67 mu m(2), respectively, at 3 d. Microvilli structural gene expression portrayed diverse patterns. Expression of Plastin 1, which bundles and binds actin cores to the terminal web, increased 8.7-fold between 17 E and DOH (P = 0.005), and gradually increased up to 7 d (P = 0.045). Ezrin and Myo1a, both actin core-cell membrane cross-linkers, portrayed dif-ferent expression patterns throughout the perihatch period, as Ezrin expression was relatively stable, while Myo1a expression increased 15.8-fold between 17 E and 10 d (P < 0.0001). We conclude that microvilli assem -bly during the perihatch period is a rapid, coordinated process, which dramatically expands the digestive and absorptive surface area of the small intestine before the completion of villi maturation.
The marketplace was the central hub for economic activity in the medieval city. Among its important functions was the provision of an open, visible space within which transactions were subject to official and communal oversight, thus according them legitimacy. This article examines the validating space created by the marketplace with respect to Jewish-Christian economic interactions. The reliance on spatial divides in Jewish-Christian economic exchange is explored by examining the local variations of the Jewish trade privilege, which allowed Jews in the German Empire to receive compensation for stolen items found in their possession. While the public space of the city initially provided Jews with protection regarding this privilege, later in the 13th century the privilege could not be applied once goods were exposed outside of Jews' homes. The changing attitudes and approaches toward Jewish economic activity are traced by contextualizing local legislation from the German Empire during the 13th century with contemporaneous responsa literature.
Aims: Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies, systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers, and as such qualify the method as local inflammation model for clinical pharmacology studies.
Methods: Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion, skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate.
Results: LPS elicited a visible response and returned to baseline at 48 hours. Erythema, perfusion and temperature were statistically significant (P < .0001) over a 24-hour time course compared to saline. The protein response was dominated by an acute interleukin (IL)-6, IL-8 and tumour necrosis factor response followed by IL-1β, IL-10 and interferon-γ. The cellular response consisted of an acute neutrophil influx followed by different monocyte subsets and dendritic cells.
Discussion: Intradermal LPS administration in humans causes an acute, localized and transient inflammatory reaction that is well-tolerated by healthy volunteers. This may be a valuable inflammation model for evaluating the pharmacological activity of anti-inflammatory investigational compounds in proof of pharmacology studies.
We study the representation of attitudes to risk in Jeffrey’s (The logic of decision, University of Chicago Press, Chicago, 1965) decision-theoretic framework suggested by Stefánsson and Bradley (Philos Sci 82(4):602–625, 2015; Br J Philos Sci 70(1):77–102, 2017) and Bradley (Econ Philos 32(2):231–248, 2016; Decisions theory with a human face, Cambridge University Press, Cambridge, 2017). We show that on this representation, the value of any prospect may be expressed as a sum of two components, the prospect’s instrumental value (the value the prospect has only in virtue of the outcomes it might lead to) and the prospect’s intrinsic value (the value the prospect has only in virtue of the way it assigns different probabilities to the different outcomes). Both components have an expectational form. We also make a distinction between a prospect’s overall intrinsic value and a prospect’s conditional intrinsic value given each one of its possible outcomes and argue that this distinction has great explanatory power. We explore the relation between these two types of intrinsic values and show that they are determined at the level of preferences. Finally, we explore the relation between the intrinsic values of different prospects and point to a strong restriction on this relation that is implicit in Jeffrey’s axioms. We suggest a natural interpretation to this restriction.
