The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.

BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)-A Novel Approach to Characterize Protein-Protein Interactions in Living Cells by Similar Isothermal Dose-Responses.

Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose-response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein-protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose-responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose-response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named "small molecule arranged thermal proximity coaggregation" (smarTPCA).

Comparative Secretomics Gives Access to High Confident Secretome Data: Evaluation of Different Methods for the Determination of Bona Fide Secreted Proteins.

Secretome analysis is broadly applied to understand the interplay between cells and their microenvironment. In particular, the unbiased analysis by mass spectrometry-based proteomics of conditioned medium has been successfully applied. In this context, several approaches have been developed allowing to distinguish proteins actively secreted by cells from proteins derived from culture medium or proteins released from dying cells. Here, three different methods comparing conditioned medium and lysate by quantitative mass spectrometry-based proteomics to identify bona fide secreted proteins are evaluated. Evaluation in three different human cell lines reveals that all three methods give access to a similar set of bona fide secreted proteins covering a broad abundance range. In the analyzed primary cells, that is, mesenchymal stromal cells and normal human dermal fibroblasts, more than 70% of the identified proteins are linked to classical secretion pathways. Furthermore, 4-12% are predicted to be released by unconventional secretion pathways. Interestingly, evidence of release by ectodomain shedding in a large number of the remaining candidate proteins is found. In summary, it is convinced that comparative secretomics is currently the method of choice to obtain high-confident secretome data and to identify novel candidates for unconventional protein secretion which have been neglected so far.

Why flipping the classroom is not enough: Digital curriculum making after the pandemic.

To slow down the proliferation of Covid-19, governments virtually shut down public life, temporarily closed schools, and forced teaching to be done exclusively on a remote basis. These measures offer an opportunity to reexamine conventional teaching and learning arrangements, test new digital and analogue concepts, and provide essential inspiration for curriculum making in the twenty-first century. This article addresses the historical development of schooling in the classroom as differentiated from "homeschooling". On one hand, the question of how school closures and digitally supported teaching settings may affect an increase in educational inequalities is investigated using an international comparison. On the other hand, the pedagogical and didactical implications of distance learning and a digital teaching culture, which constitute the foundation for digital curriculum making, are examined.

The negative piezoelectric effect of the ferroelectric polymer poly(vinylidene fluoride).

Piezoelectricity describes interconversion between electrical charge and mechanical strain. As expected for lattice ions displaced in an electric field, the proportionality constant is positive for all piezoelectric materials. The exceptions are poly(vinylidene fluoride) (PVDF) and its copolymers with trifluoroethylene (P(VDF-TrFE)), which exhibit a negative longitudinal piezoelectric coefficient. Reported explanations exclusively consider contraction with applied electric field of either the crystalline or the amorphous part of these semi-crystalline polymers. To distinguish between these conflicting interpretations, we have performed in situ dynamic X-ray diffraction measurements on P(VDF-TrFE) capacitors. We find that the piezoelectric effect is dominated by the change in lattice constant but, surprisingly, it cannot be accounted for by the polarization-biased electrostrictive contribution of the crystalline part alone. Our quantitative analysis shows that an additional contribution is operative, which we argue is due to an electromechanical coupling between the intermixed crystalline lamellae and amorphous regions. Our findings tie the counterintuitive negative piezoelectric response of PVDF and its copolymers to the dynamics of their composite microstructure.

Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins.

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 µM, 6.0 ± 2.9 µM, and 10.06 ± 2.87 µM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.

Comparison of cardiovascular risk calculation tools in pharmacy practice.

OBJECTIVE: To examine the use of various cardiovascular disease (CVD) risk estimation calculators in pharmacy practice. DESIGN: Longitudinal cohort study. SETTING: Midwestern university worksite from August 2008 through May 2012. PARTICIPANTS: University employees with hypertension, dyslipidemia, and diabetes. INTERVENTION: Risk estimation calculators were applied to data from a pharmacist-run chronic disease management program. MAIN OUTCOME MEASURE: Difference in estimated CVD risk from multiple estimation calculators. RESULTS: At baseline and 12 months, non-lab-based tools reported significantly higher 10-year CVD risk percentages compared with lab-based tools among the same cohort of patients (10.63% vs. 8.71% at baseline, P < 0.001; 9.34% vs. 7.31% at 12 months, P < 0.001). In addition, the electronic version of 10-year CVD risk reported significantly higher values than the paper version when applied to the same patient cohort (7.31% vs. 6.60% at 12 months, P = 0.018). CONCLUSION: CVD risk estimation tools report significantly different values and are not interchangeable. Pharmacists using non-lab-based tools should expect significantly higher risk estimates than estimates derived from lab-based tools and therefore should use the same version of the estimation tool over the long term.

Using employee experts to offer an interprofessional diabetes risk reduction program to fellow employees.

A recent increase in the incidence of diabetes and pre-diabetes is causing many employers to spend more of their healthcare benefit budgets to manage the conditions. A self-insured university in the USA has implemented an interprofessional diabetes mellitus risk reduction program using its own employee faculty and staff experts to help fellow employees manage their diabetes and pre-diabetes. The interprofessional team consists of five pharmacists, a dietitian, an exercise physiologist, a health educator and a licensed mental health practitioner. In addition, the participant's physician serves as a consultant to the program, as does a human resources healthcare benefits specialist and a wellness coordinator. The volunteer program takes place at the worksite during regular business hours and is free of charge to the employees. The faculty and staff delivering the program justify the cost of their time through an interprofessional educational model that the program will soon provide to university students.

Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells.

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and ß) in sustaining malignant cells' growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90ß isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.

Self-assembled monolayer exchange reactions as a tool for channel interface engineering in low-voltage organic thin-film transistors.

In this work, we compared the kinetics of monolayer self-assembly long-chained carboxylic acids and phosphonic acids on thin aluminum oxide surfaces and investigated their dielectric properties in capacitors and low-voltage organic thin-film transistors. Phosphonic acid anchor groups tend to substitute carboxylic acid molecules on aluminum oxide surfaces and thus allow the formation of mixed or fully exchanged monolayers. With different alkyl chain substituents (n-alkyl or fluorinated alkyl chains), the exchange reaction can be monitored as a function of time by static contact angle measurements. The threshold voltage in α,α'-dihexyl-sexithiophene thin-film transistors composed of such mixed layer dielectrics correlates with the exchange progress and can be tuned from negative to positive values or vice versa depending on the dipole moment of the alkyl chain substituents. The change in the dipole moment with increasing exchange time also shifts the capacitance of these devices. The rate constants for exchange reactions determined by the time-dependent shift of static contact angle, threshold voltage, and capacitance exhibit virtually the same value thus proving the exchange kinetics to be highly controllable. In general, the exchange approach is a powerful tool in interface engineering, displaying a great potential for tailoring of device characteristics.

Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe.

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.

Milmed Yeast Alters the LPS-Induced M1 Microglia Cells to Form M2 Anti-Inflammatory Phenotype.

Microglial cells polarized towards a proinflammatory phenotype are considered the main cellular players of neuroinflammation, underlying several neurodegenerative diseases. Many studies have suggested that imbalance of the gut microbial composition is associated with an increase in the pro-inflammatory cytokines and oxidative stress that underlie chronic neuroinflammatory diseases, and perturbations to the gut microbiota were detected in neurodegenerative conditions such as Parkinson's disease and Alzheimer's disease. The importance of gut-brain axis has been uncovered and the relevance of an appropriate microbiota balance has been highlighted. Probiotic treatment, rebalancing the gut microbioma, may reduce inflammation. We show that Milmed yeast, obtained from S. cerevisiae after exposure to electromagnetic millimeter wavelengths, induces a reversal of LPS-M1 polarized microglia towards an anti-inflammatory phenotype, as demonstrated morphologically by the recovery of resting phenotype by microglia, by the decrease in the mRNAs of IL-1ß, IL-6, TNF-α and in the expression of iNOS. Moreover, Milmed stimulated the secretion of IL-10 and the expression of Arginase-1, cell markers of M2 anti-inflammatory polarized cells. The present findings data suggest that Milmed may be considered to be a probiotic with diversified anti-inflammatory activity, capable of directing the polarization of microglial cells.

Solid-Phase Synthesis of Cereblon-Recruiting Selective Histone Deacetylase 6 Degraders (HDAC6 PROTACs) with Antileukemic Activity.

In this work, we utilized the proteolysis targeting chimera (PROTAC) technology to achieve the chemical knock-down of histone deacetylase 6 (HDAC6). Two series of cereblon-recruiting PROTACs were synthesized via a solid-phase parallel synthesis approach, which allowed the rapid preparation of two HDAC6 degrader mini libraries. The PROTACs were either based on an unselective vorinostat-like HDAC ligand or derived from a selective HDAC6 inhibitor. Notably, both PROTAC series demonstrated selective degradation of HDAC6 in leukemia cell lines. The best degraders from each series (denoted A6 and B4) were capable of degrading HDAC6 via ternary complex formation and the ubiquitin-proteasome pathway, with DC50 values of 3.5 and 19.4 nM, respectively. PROTAC A6 demonstrated promising antiproliferative activity via inducing apoptosis in myeloid leukemia cell lines. These findings highlight the potential of this series of degraders as effective pharmacological tools for the targeted degradation of HDAC6.

The mycotoxin viriditoxin induces leukemia- and lymphoma-specific apoptosis by targeting mitochondrial metabolism.

Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

Implementing lifestyle medicine with medication therapy management services to improve patient-centered health care.

OBJECTIVE: To describe a patient-centered medication therapy management (MTM) program that focuses on lifestyle medicine. SETTING: Community pharmacy in Omaha, NE, from August 2008 to September 2010. PRACTICE DESCRIPTION: Traditional MTM services are combined with lifestyle medicine interventions for employees of a self-insured organization who have dyslipidemia, hypertension, and/or diabetes. Program participants meet one-on-one with a pharmacist 12 times during the first year of the program to ensure proper drug therapy and modify lifestyle behaviors (physical activity, nutrition, weight control, sleep, stress, and alcohol and tobacco use) through individualized programming. PRACTICE INNOVATION: Several patient-centered activities have been developed for the program with an emphasis on modifying lifestyle behaviors in conjunction with medications to manage participants' chronic condition. In addition, a new specialty position in health care is being developed (the ambulatist) that focuses on maintaining the ambulatory status of individuals with chronic medical conditions through appropriate drug therapy, lifestyle medicine, and care coordination. MAIN OUTCOME MEASURES: Biometric data collection and participant survey data at baseline and after 12 months. RESULTS: Pilot data for 15 participants showed improvements in all measurements, including blood cholesterol, low-density lipoprotein cholesterol, blood glucose, body weight, physical activity level, fruit and vegetable intake, risk for myocardial infarction, risk for any cardiovascular disease event, self-reported unhealthy days, and qualitative survey data. CONCLUSION: Pharmacists are in an ideal position to implement lifestyle medicine strategies in combination with MTM services to enhance patient-centered health care in a community pharmacy setting.

Pay-for-performance model of medication therapy management in pharmacy practice.

OBJECTIVE: To use an existing pharmacist-run medication therapy management (MTM)/lifestyle medicine program to propose a new model of reimbursement for pharmacists that is based on pay for performance (P4P) rather than product-based dispensing or fee for service. DATA SOURCES: Specific patient outcomes were collected during a 1-year period from an existing pharmacist-run MTM/lifestyle medicine program as the basis to propose this new model of reimbursement. DATA SYNTHESIS: The proposed model outlines a P4P model of reimbursement for pharmacists that includes both traditional MTM services and patient-centered lifestyle medicine programming. The model uses an all-or-none bundled approach for reimbursement in which the pharmacist is reimbursed at a higher rate if patients achieve all six proposed outcome criteria at 1 year. Pharmacists could earn as much a 43% more income with this model compared with traditional MTM services. This model is an incentive for payers because it is based on patient outcomes and preestablished return on investment models. CONCLUSION: Pharmacist should begin to explore ways they can participate in a high-performance health care system by moving to a P4P model of reimbursement rather than fee-for-service or product-based dispensing reimbursement models. P4P models of reimbursement could be beneficial to the patient, the payer, and the pharmacist.

Synthesis of S-adenosyl-L-homocysteine capture compounds for selective photoinduced isolation of methyltransferases.

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Using the SMART-EST Goals in Lifestyle Medicine Prescription.

Lifestyle modifications can effectively decrease chronic disease risk but studies show little to no time during patient encounters is spent on lifestyle medicine counseling. The SMART-EST goal framework facilitates both a rich discussion of lifestyle medicine and a comprehensive patient-centered action plan for health behavior change. The tenets of the SMART-EST goal-setting process are discussed.

Laying an Early Foundation: Lifestyle Medicine Pre-Professional Education (LMPP) Member Interest Group.

Just as lifestyle medicine is the necessary foundation for true health care reform, lifestyle medicine competencies should be the foundation for health education. Although lifestyle medicine education may benefit a health professional at any stage in their education or career, evidence-based undergraduate lifestyle medicine education for future health professionals shifts the perspective of health and health care delivery. Educating health preprofessionals in associate, bachelor's, master's, and other preprofessional healthcare training programs is of paramount importance due to the interdisciplinary nature of lifestyle medicine. To accomplish this, American College of Lifestyle Medicine (ACLM) members can work collaboratively through committees, projects, and working groups-becoming leadership champions of change. An ACLM Pre-Professional Member Interest Group (LMPP) was created in 2018. LMPP has been working to build a national collaborative effort to amass, create, and distribute resources for educators in this pre-professional arena. Educating college students planning to become professionals outside the medical sphere, for example, lawyers, business people, artists, and engineers, will also benefit the field by introducing the power of nutrition, exercise, sleep, social connection, and stress resiliency during this formative state of career development. Pre-professional educational programs provide learners the opportunity to personally experience the power of lifestyle medicine.