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In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3∆ mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling., (Copyright © 2022 Elsevier B.V. All rights reserved.)

BPMN Sketch Miner is a modeling environment for generating visual business process models starting from constrained natural language textual input. Its purpose is to support business process modelers who need to rapidly sketch visual BPMN models during interviews and design workshops, where participants should not only provide input but also give feedback on whether the sketched visual model represents accurately what has been described during the discussion. In this article, we present a detailed description of the BPMN Sketch Miner design decisions and list the different control flow patterns supported by the current version of its textual DSL. We also summarize the user study and survey results originally published in MODELS 2020 concerning the tool usability and learnability and present a new performance evaluation regarding the visual model generation pipeline under actual usage conditions. The goal is to determine whether it can support a rapid model editing cycle, with live synchronization between the textual description and the visual model. This study is based on a benchmark including a large number of models (1350 models) exported by users of the tool during the year 2020. The main results indicate that the performance is sufficient for a smooth live modeling user experience and that the end-to-end execution time of the text-to-model-to-visual pipeline grows linearly with the model size, up to the largest models (with 195 lines of textual description) found in the benchmark workload., (© The Author(s) 2022.)

In response to environmental changes cells rapidly rearrange their gene expression pattern in order to adapt to the new conditions. Chromatin remodeling is critical for this process playing a major role in the induction of genes involved in stress responses. We demonstrated previously that TPK1, encoding one of the catalytic subunits of PKA from Saccharomyces cerevisiae, is upregulated under heat shock. Herein, we investigate the chromatin remodeling of the TPK1, TPK2 and TPK3 promoters under heat stress. The TPK1 promoter is the only one that presents three positioned nucleosomes. Upon heat stress or osmostress these nucleosomes are evicted in clear correlation with promoter activation and upregulation of TPK1 mRNA levels. We find that remodelers SWI/SNF, RSC, INO80 and ISW1 participate in chromatin remodeling of the TPK1 promoter under thermal stress conditions. RSC and INO80 are necessary for nucleosomes positioning and contribute to repression of the TPK1 promoter under normal conditions while SWI/SNF participates in the eviction of nucleosomes after heat stress. SWI/SNF complex is recruited to the TPK1 promoter upon heat shock in a Msn2/4-dependent manner. Finally, both Tpk1 and Tpk2 catalytic subunits are recruited to the TPK1 promoter with opposite association patterns. Tpk1 catalytic activity is necessary for nucleosome rearrangement on the TPK1 promoter while Tpk2 and Tpk3 inhibit the promoter activity and maintain a repressive chromatin conformation. This work enlightens the mechanism of regulation of TPK1 expression during heat-stress, contributing to the knowledge of specificity in fine-tuning the cAMP-PKA signaling circuit., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: multiple myeloma (MM) is a plasma cell disorder that represents the second most frequent hematologic cancer. Although MM is still an incurable disease, prognosis has improved in the last decades thanks to the introduction of novel agents such as proteasome inhibitors (PIs), immunomodulatory drugs, monoclonal antibodies, and histone deacetylase inhibitors. Areas covered: ixazomib is the first oral PI recently approved by Food and Drug Administration (FDA) and European Medicine Agency (EMA) in combination with lenalidomide and dexamethasone as salvage therapy in MM patients. In this paper, we focus on its pharmacokinetics features, as well as its safety and efficacy in clinical studies. Expert opinion: ixazomib can be considered an oral analogue of bortezomib, with 9.5-day half-life, 58% of oral bioavailability, and a large distribution volume of 543L. These features make it a versatile molecule, potentially useful both in combination and as single agent. Oral route of administration and good efficacy/safety profile are its winning characteristics, providing the rationale for a future role also in the maintenance setting.

Yeast cells can adapt their growth in response to the nutritional environment. Glucose is the favourite carbon source of Saccharomyces cerevisiae, which prefers a fermentative metabolism despite the presence of oxygen. When glucose is consumed, the cell switches to the aerobic metabolism of ethanol, during the so-called diauxic shift. The difference between fermentative and aerobic growth is in part mediated by a regulatory mechanism called glucose repression. During glucose derepression a profound gene transcriptional reprogramming occurs and genes involved in the utilization of alternative carbon sources are expressed. Protein kinase A (PKA) controls different physiological responses following the increment of cAMP as a consequence of a particular stimulus. cAMP-PKA is one of the major pathways involved in the transduction of glucose signalling. In this work the regulation of the promoters of the PKA subunits during respiratory and fermentative metabolism are studied. It is demonstrated that all these promoters are upregulated in the presence of glycerol as carbon source through the Snf1/Cat8 pathway. However, in the presence of glucose as carbon source, the regulation of each PKA promoter subunits is different and only TPK1 is repressed by the complex Hxk2/Mig1 in the presence of active Snf1. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Introduction: Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell proliferation and overproduction of monoclonal paraprotein, hypercalcemia, renal failure, anemia, osteolytic bone lesions, and infections. Melphalan, a nitrogen mustard, is an alkylating agent synthesized in 1953, and it has been used in multiple myeloma therapy for fifty years. Although novel agents have been introduced in the past few decades improving prognosis of the disease, melphalan still maintains a crucial role in the treatment of MM acting both as cytotoxic agent through damage to DNA, and as immunostimulatory drug by inhibiting Interleukin-6, as well as interaction with dendritic cells, and immunogenic effects in tumor microenvironment. Areas covered: This review focuses on available data about melphalan pharmacology and its role in clinical practice. Expert opinion: Melphalan remains crucial in therapy of multiple myeloma because of its good manageability, safety profile, efficacy, and economic sustainability. These characteristics make it pivotal also for new regimens in combination with novel agents.

We analyzed 50 patients who achieved at least a very good partial response in the RV-MM-EMN-441 study. Patients received consolidation with autologous stem-cell transplantation (ASCT) or cyclophosphamide-lenalidomide-dexamethasone (CRD), followed by Lenalidomide-based maintenance. We assessed minimal residual disease (MRD) by multi-parameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) after consolidation, after 3 and 6 courses of maintenance, and thereafter every 6 months until progression. By MFC analysis, 19/50 patients achieved complete response (CR) after consolidation, and 7 additional patients during maintenance. A molecular marker was identified in 25/50 patients, 4/25 achieved molecular-CR after consolidation, and 3 additional patients during maintenance. A lower MRD value by MFC was found in ASCT patients compared with CRD patients (p=0.0134). Tumor burden reduction was different in patients with high-risk vs standard-risk cytogenetics (3.4 vs 5.2, ln-MFC; 3 vs 6 ln-PCR, respectively) and in patients who relapsed vs those who did not (4 vs 5, ln-MFC; 4.4 vs 7.8 ln-PCR). MRD progression anticipated clinical relapse by a median of 9 months while biochemical relapse by a median of 4 months. MRD allows the identification of a low-risk group, independently of response, and a better characterization of the activity of treatments.

The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives/background: We herein investigated the role of polymorphisms in calcitonin gene-related peptide (CGRP)-related genes looking at the association of rs3781719 (T > C) in the calcitonin gene-related polypeptide-alpha (CALCA) gene and of rs3754701 (T > A) and rs7590387 (C > G) at the receptor activity modifying 1 (RAMP1) locus with triptan response in patients with migraine without aura (MwoA). In addition, their role was evaluated as risk factors for transformation of episodic migraine into medication overuse headache (MOH). The CGRP has a central role in the pathogenesis of migraine; however, little information is currently available concerning the role of polymorphisms in CGRP-related genes as determinants of clinical response to anti-migraine drugs or as risk factors for migraine chronification., Methods: Genotyping was conducted retrospectively by real-time polymerase chain reaction allelic discrimination assay in 219 patients with MwoA and 130 with MOH in whom migraine was the primary headache type. Gene variants association was evaluated by logistic regression analysis adjusted by confounding factors. The threshold of statistical significance was set according to the total number of polymorphisms analyzed in the current study and in previous publications arising from overlapping datasets., Results: No evidence of association was found between the three polymorphisms tested and triptan response in MwoA patients. Conversely, carriers of RAMP1 rs7590387GG displayed a lower risk of episodic migraine transformation into MOH (vs C allele carriers, odds ratio [OR]: 0.27, 95% confidence interval [CI]: 0.13-0.57, P = 0.0002; threshold of significance set at P < 0.0029). When genotype distribution for RAMP1 rs7590387 was compared between healthy controls (n = 209) and MOH patients, carriers of rs7590387GG were found at lower risk of developing MOH (OR: 0.43, 95%CI: 0.22-0.85, P = 0.011)., Conclusion: These results suggest that RAMP1 rs7590387 may have a role in the transformation of episodic migraine into MOH., (© 2015 American Headache Society.)

Introduction: The 26S proteasome has many important roles in the biological functions of the cells, and proteasome inhibitors have multiple and complex activities on cells. These compounds can be natural or synthesized. Most synthetic derivatives have been rationally designed, synthesized and optimized to obtain the best selectivity and increase the activity. The design of chemical entities with desired molecular identification, which plays an important role in biological systems, is provided by pharmacophore modeling. Indeed, pharmacophore models can be established either in a ligand-based manner or in a receptor-based manner., Areas Covered: The authors discuss the application of pharmacophore modeling techniques to proteasome inhibitors development. Furthermore, the article reviews the classification of the currently discovered proteasome inhibitors where the principal mechanism of action and clinical application are represented., Expert Opinion: In the era of new drug development, database of compounds should be thoroughly evaluated with a combination of methods that consider both pharmacophore- and ligand-based virtual screening. The concept of pharmacophore helps to discover new active compounds and to evaluate their activity. The nature of proteasome inhibitor pharmacophore affects the secondary active-site specificity; indeed, increasing specificity decreases the cytotoxicity of the proteasome inhibitors. It is hypothesized that the balanced simultaneous modulation of a few druggable targets may have superior efficacy and fewer side effects than single-target or combination therapies for the treatment of human cancers. The discovery of new compounds should aim to find more active compounds that improve the compliance of patients.

Despite the improvements thanks to the introduction of proteasome inhibitors and immunomodulatory drugs (IMiDs), nearly all myeloma patients eventually become refractory to these drugs. Consequently, the outcome of these patients is very poor. Pomalidomide is a new IMiD with a similar structure to the commonly used IMiD thalidomide and lenalidomide. Pomalidomide exhibited more potent anti-myeloma activity and a similar favorable safety profile compared with thalidomide and lenalidomide. In Phase I-II studies pomalidomide plus low-dose dexamethasone demonstrated activity in myeloma patients refractory to both bortezomib and IMiDs. Based on the results of a Phase III trial, the FDA and EMA agencies granted accelerated approval to pomalidomide, which is now considered a new effective strategy for relapsed and/or refractory myeloma patients. Very promising results were obtained when pomalidomide-dexamethasone was used in combination with other compounds. This review provides updated information about pharmacokinetics, mechanism of action, resistance, clinical efficacy and safety of pomalidomide.

Protein kinase A (PKA) is a broad specificity protein kinase that controls a physiological response following the increment of cAMP as a consequence of a particular stimulus. The specificity of cAMP-signal transduction is maintained by several levels of control acting all together. Herein we present the study of the regulation of the expression of each PKA subunit, analyzing the activity of their promoters. The promoter of each isoform of TPK and of BCY1 is differentially activated during the growth phase. A negative mechanism of isoform-dependent autoregulation directs TPKs and BCY1 gene expressions. TPK1 promoter activity is positively regulated during heat shock and saline stress. The kinase Rim15, but not the kinase Yak1, positively regulates TPK1 promoter. Msn2/4, Gis1, and Sok2 are transcription factors involved in the regulation of TPK1 expression during stress. TPK2, TPK3, and BCY1 promoters, unlike TPK1, are not activated under stress conditions, although all the promoters are activated under low or null protein kinase A activity. These results indicate that subunits share an inhibitory autoregulatory mechanism but have different mechanisms involved in response to heat shock or saline stress., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Introduction: The development of novel agents, such as immunomodulatory drugs and proteasome inhibitors, has led to a considerable increment in the response rate (RR) and outcomes for multiple myeloma (MM) patients. Unfortunately, MM patients will inevitably relapse and become resistant to new drugs. This led to the continuous development of novel agents. Carfilzomib is a second-generation proteasome inhibitor, demonstrating promising results in relapsed/refractory (RR) and newly diagnosed (ND) MM patients., Areas Covered: Herein, the authors review Phase I and II trials on carfilzomib for the treatment of MM. They also describe the profile of the drug during Phase I escalating doses and evaluate the efficacy of carfilzomib both alone and in combination. Finally, the authors also review and discuss the carfilzomib safety profile., Expert Opinion: Clinical trials (Phases I and II) with carfilzomib, used both as single agent or in combination with other therapies, established the maximum tolerated dose and recommended schedule of administration. Preliminary data showed that it had a high efficacy and a good safety profile both in RRMM and NDMM patients. Carfilzomib seems to be effective in patients previously treated with bortezomib. Future Phase II and III studies will better define the role of carfilzomib in the treatment of MM as well as its optimum dose.

Introduction: Lenalidomide is an oral immunomodulatory drug, which was recently introduced for the treatment of multiple myeloma (MM). It has been used for the treatment of newly diagnosed and relapsed MM patients, as both maintenance and preventive therapy. Available data show a progression-free survival and overall survival improvement associated with this drug., Areas Covered: Efficacy results of lenalidomide in Phase I, II, and III trials in MM are reported herein. The recent use of lenalidomide as maintenance and preventive therapy is described. An overview of the most important adverse events is also presented, such as myelosuppression, thromboembolic events, fatigue, dermatologic toxicity, infections, teratogenic potential, and second primary malignancies. The literature reviewed consists of clinical trials published from 2001 to 2012., Expert Opinion: Lenalidomide is one of the most important therapeutic agents for MM treatment. Various trials have confirmed its remarkable anticancer activity, alone or combined with high- or low-dose dexamethasone, or other drugs. Lenalidomide-related adverse events can be controlled with dose reductions, supportive therapy, and appropriate prophylaxis.

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10-50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005). The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.

Biomarker detection is one of the greatest challenges in Clinical Proteomics. Today, great hopes are placed into tandem mass spectrometry (MS/MS) to discover potential biomarkers. MS/MS is a technique that allows large scale data analysis, including the identification, characterization, and quantification of molecules. Especially the identification process, that implies to compare experimental spectra with theoretical amino acid sequences stored in specialized databases, has been subject for extensive research in bioinformatics since many years. Dozens of identification programs have been developed addressing different aspects of the identification process but in general, clinicians are only using a single tools for their data analysis along with a single set of specific parameters. Hence, a significant proportion of the experimental spectra do not lead to a confident identification score due to inappropriate parameters or scoring schemes of the applied analysis software. The swissPIT (Swiss Protein Identification Toolbox) project was initiated to provide the scientific community with an expandable multi-tool platform for automated and in-depth analysis of mass spectrometry data. The swissPIT uses multiple identification tools to automatic analyze mass spectra. The tools are concatenated as analysis workflows. In order to realize these calculation-intensive workflows we are using the Swiss Bio Grid infrastructure. A first version of the web-based front-end is available (http://www.swisspit.cscs.ch) and can be freely accessed after requesting an account. The source code of the project will be also made available in near future.