Structural interpretation of the amino acid sequence of a second domain from the Artemia covalent polymer globin

Artemia has a complex extracellular hemoglobin of Mr 260,000 comprising two globin chains (Mr 130,000) each of which is a polymer of eight covalently linked domains of Mr 16,000. The primary structure of this polymeric globin was studied to understand how globin folded domains are ordered within a globin chain and, in turn, how the latter associate into a functional hemoglobin molecule. Here we report the amino acid sequence of a second domain, E7 (Mr 16,081, excluding the heme), and interpretations of sequence data by computer-assisted alignment and modeling. This clearly shows that, as with domain E1 (Moens, L. Van Hauwaert, M.-L. De Smet, K. Geelen, D. Verpooten, G. Van Beeumen, J. Wodak, S. Alard, P. & Trotman, C. (1988) J. Biol. Chem. 263, 4679-4685), domain E7 is compatible with a globin folded structure of the β-type chain. Several specific differences of domains E7 and E1 from the classic globins are identified. They possibly can be interpreted in terms of specific requirements for a double octameric functional molecule.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Adult rabbit cardiomyocytes undergo typical hibernation-like dedifferentiation when co-cultured with cardiac fibroblasts.

Objectives: Little is known about the causal factors which induce the typical structural changes accompanying cardiomyocyte dedifferentiation in vivo such as in chronic hibernating myocardium. For identifying important factors involved in cardiomyocyte dedifferentiation, as seen in chronic hibernation, an in vitro model mimicking those morphological changes, would be extremely helpful. Methods: Adult rabbit cardiomyocytes were co-cultured with cardiac fibroblasts. The typical changes induced by this culturing paradigm were investigated using morphometry, electron microscopy and immunocytochemical analysis of several structural proteins, which were used as dedifferentiation markers, i.e., titin, desmin, cardiotin and alpha -smooth muscle actin. Results: Close apposition of fibroblasts with adult rabbit cardiomyocytes induced hibernation-like dedifferentiation, similar to the typical changes seen in chronic hibernation in vivo. Both changes in ultrastructure and in the protein expression pattern of dedifferentiation markers as seen in chronic hibernating myocardium were seen in the co-cultured cardiomyocytes. Conclusion: Hibernation-like changes can be induced by co-culturing adult rabbit cardiomyocytes with fibroblasts. This cellular model can be a valuable tool in identifying and characterizing the pathways involved in the dedifferentiation phenotype in vivo, and already suggests that many of the structural changes accompanying dedifferentiation are not per se dependent on a decreased oxygen availability

Panic results in unique molecular and network changes in the amygdala that facilitate fear responses

Recurrent panic attacks (PAs) are a common feature of panic disorder (PD) and post-traumatic stress disorder (PTSD). Several distinct brain regions are involved in the regulation of panic responses, such as perifornical hypothalamus (PeF), periaqueductal grey, amygdala and frontal cortex. We have previously shown that inhibition of GABA synthesis in the PeF produces panic-vulnerable rats. Here, we investigate the mechanisms by which a panic-vulnerable state could lead to persistent fear. We first show that optogenetic activation of glutamatergic terminals from the PeF to the basolateral amygdala (BLA) enhanced the acquisition, delayed the extinction and induced the persistence of fear responses 3 weeks later, confirming a functional PeF-amygdala pathway involved in fear learning. Similar to optogenetic activation of PeF, panic-prone rats also exhibited delayed extinction. Next, we demonstrate that panic-prone rats had altered inhibitory and enhanced excitatory synaptic transmission of the principal neurons, and reduced protein levels of metabotropic glutamate type 2 receptor (mGluR2) in the BLA. Application of an mGluR2 positive allosteric modulator (PAM) reduced glutamate neurotransmission in the BLA slices from panic-prone rats. Treating panic-prone rats with mGluR2 PAM blocked sodium lactate (NaLac)-induced panic responses and normalized fear extinction deficits. Finally, in a subset of patients with comorbid PD, treatment with mGluR2 PAM resulted in complete remission of panic symptoms. These data demonstrate that a panic-prone state leads to specific reduction in mGluR2 function within the amygdala network and facilitates fear, and mGluR2 PAMs could be a targeted treatment for panic symptoms in PD and PTSD patients

Selective inhibition of intestinal guanosine 3,5-cyclic monophosphate signaling by small-molecule protein kinase inhibitors

The guanosine 3,5-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the C-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII

Developing a patient decision aid to improve shared decision making in breast cancer.

During the last two decades, shared decision making (SDM) has been increasingly applied in multiple health care decision contexts. Within the model of SDM, a patient and physician share information regarding the benefits and risks of potential options and personal preferences. They discuss the patient's preferences for each of the available options and discuss the patient's desire for involvement in decision making. Finally, they make or defer a decision and arrange follow-up if applicable. SDM is the preferred model for medical decision making in case of preference-sensitive decisions. Within these decisions, two or more options are available, none of which is clearly superior. To facilitate the process of SDM, patient decision aids (PtDAs) can be applied. The aim of a PtDA is threefold: they make the decision explicit and provide evidence-based information on the potential options, they help patients clarify their values and preferences for the different options and they support patients to communicate their preferences to their health care provider. PtDAs exist in various formats, ranging from paper-based brochures to interactive digital applications, using audio and video elements. The International Patient Decision Aids Standards Collaboration has established a checklist for the development of PtDAs, based on 12 quality domains, covering content, development process and effectiveness of the PtDA. Internationally developed PtDAs have proven to increase patients' knowledge and accuracy of risk perception, and decrease their decisional conflict. There are multiple PtDAs available in the disease areas of breast, prostate and lung cancer, osteoarthritis and osteoporosis, end of life decisions and cholesterol-related diseases. The variety in disease areas highlights that preference-sensitive decisions occur in many different health care decision contexts. The disease area of hormone-sensitive breast cancer is particularly suitable for the implementation of a PtDA. Patients receiving adjuvant endocrine therapy undergo a very long treatment that often causes a high impact on quality of life, resulting in suboptimal treatment adherence. The available treatments, tamoxifen or aromatase inhibitors, may cause adverse events such as increased risk of blood clots or joint and muscle pain, respectively. The efficacy of both treatments is comparable, although aromatase inhibitors are considered to be the most effective option. As treatment impact varies considerably between patients, a PtDA can help to tradeoff potential benefits and adverse events to determine the optimal therapy for individual patients. Although the practice of SDM and the use of PtDAs is internationally recognized, there are currently only very few PtDAs available in Belgium and there are no examples available for patients with breast cancer. This PhD project aimed to improve SDM for Belgian patients with hormone-sensitive breast cancer by developing a PtDA for decisions regarding switching or continuing adjuvant endocrine therapy after 2-3 years. This PtDA should meet the needs of both patient and physician, by informing patients on the available decision options, eliciting their preferences for these options and supporting them to communicate their preferences to their treating physician. In order to meet this aim, four specific objectives were identified. The first objective was to compare the process of decision making between patients and consumers and to identify innovative aspects of preference elicitation methods from the consumer research field. A literature review showed that the decision process between patients and consumers is highly comparable and identified five concepts from the consumer research field that might improve preference elicitation in healthcare. Preference elicitation methods that resemble real-life decision making as closely as possible, for example by providing time for self-reflection, are likely to generate the most accurate results. The second objective was to assess the needs of both patients and physicians regarding a PtDA for breast cancer decision making. Four focus groups with 21 patients and five individual interviews with breast cancer specialists indicated that patients currently experience little involvement in their treatment decision making. Patients furthermore indicated a high need for information regarding treatment options, especially quantitative information on treatment benefits and risks. The breast cancer specialists acknowledged that SDM has become more important during the last decade but stated that it remains unclear how to implement this in clinical practice. Decisions regarding adjuvant endocrine therapy were deemed ideal for a PtDA intervention by both stakeholders as these treatments may have a high impact on quality of life and deliver limited benefits. Beneficial PtDA features were identified, such as the possibility to write down questions or to rate the impact of adverse events on daily life. The third objective was to develop an interactive, online PtDA for patients with hormone-sensitive breast cancer switching adjuvant endocrine treatment. The combination of an in-depth literature review and stakeholder interviews were used to determine the content and design of the PtDA. Five attribute categories were identified using this approach: efficacy, adverse events, use, impact on quality of life and mechanism of action. Both patients and physicians rated potential attributes to determine the final selection, which included breast cancer mortality, risk of recurrence, treatment duration, joint and muscle pain, osteoporosis and increased thrombosis risk. The developed prototype PtDAs consists of three consecutive modules; an information module aiming to educate patients, a scenario-based module that will help patients to clarify potential impact on their everyday life and an adaptive conjoint analysis exercise to elicit patients' preferences for various treatment characteristics. The final objective was to test the developed PtDA in a two-stage process. First, alpha testing in a research setting yielded an average usability score of 78.75 out of 100 using the System Usability Scale. Furthermore, content and lay-out were scored 8.9 and 8.5 out of 10 respectively by 11 patients; and quality, completeness and lay-out were scored 8.4, 8.4 and 8.2 out of 10 respectively by five health care professionals. Qualitative feedback was gathered by applying cognitive interviewing while using the PtDA and a short interview afterwards. Next, after implementing the feedback received during alpha-testing, beta testing in a clinical setting was performed using a pilot trial. Nine patients tested the PtDA in the week before their planned follow-up consultation at the university hospital of Leuven. The effect of the PtDA was assessed by determining the impact on the quality of the decision process and the decision itself using the following constructs: knowledge, values-choice agreement, feeling informed, feeling clear about values, discussing goals with health care providers, and being involved. Patient knowledge increased from 5.33 before using the PtDA to 7.78 afterwards. Decisional conflict was low after the consultation, with a score of 18.06. One month after the consultation, decisional conflict had increased significantly, with a score of 41,67. This might indicate the need for a broader support framework for patients by providing information and support them to discuss their preferences over a longer time period. Making the PtDA available after the consultation and planning follow-up consultation when needed could be potential solutions. The extent to which SDM was applied during the consultation was assessed using the SDM-9 item questionnaire. The average score of 20 out of a possibly 45 indicated that no real SDM took place, highlighting that patient directed interventions only, are not sufficient to implement SDM in clinical practice. Usability was again assessed using the System Usability Scale, resulting in a mean score of 71.25. Finally, patient preferences elicited for different treatment characteristics revealed high variability between patients, with a total of five different attributes being selected as 'the most important one' by seven patients. These results indicate the need to discuss individual treatment preferences during consultations. Based on the information gained through the different chapters, recommendations are formulated for the future development of PtDAs in Belgium and the implementation of SDM in clinical practice. The first set of recommendations is related to the development process of PtDAs. As the current use and development of PtDAs is very limited in Belgium, more research in other disease areas is needed to truly enable patient centered care. Stakeholder input has proven to be indispensable during the development process. Therefore, stakeholder opinions regarding PtDA design and content should be assessed using a combination of qualitative and quantitative approaches. Different formats or visual displays should be tested to meet the specific needs of the target group. Interactive, online applications offer a range of advantages regarding implementation and use. Video and audio materials can facilitate the learning process for users. Interactive features such as content control or the use of narratives may improve decision making. Furthermore, explicit preference elicitation methods that simulate real-life decision making may improve value clarification. The generated preferences weights can open up the discussion during a consultation. More research is required to facilitate optimal implementation in Belgium. The implementation strategy for a PtDA should already be considered during the development process. A centralized platform, either for the whole of Belgium or for Flanders, may provide easy access to PtDAs. However, not all patients are familiar with an online environment, nor does everyone have internet access. The needs of the target group should be assessed to identify the best implementation approach to allow for patient-centered care. The second set of recommendations is developed to improve SDM in Belgium. The main recommendation here is to create awareness for SDM and to support implementation in clinical practice. A combination of clinical guidelines and practical measures such as logistic and financial support are needed. More research is needed to identify and address the barriers of Belgian health care professionals for SDM implementation. An example can be financial support to reimburse physicians for the time spent on the conduct of true SDM, for example in analogy to the compensation pharmacists can receive for performing counseling on the use of diabetes or asthma medicines. Moreover, financial support will be required to stimulate research on the development of PtDAs and the implementation of SDM. Furthermore, initiatives from various stakeholders and organizations should be harmonized in one collective approach. We should capitalize on the knowhow built up during international research, especially from countries with a comparable health care system such as the Netherlands. Care pathways in both primary and secondary care might need to be revised, to allow patients the necessary time to inform themselves and participate in decision making. More quality measures are needed to monitor the extent of SDM in clinical practice and to improve where needed. Another important recommendation is to apply trainings in SDM for health care professionals. If we truly want to implement SDM in routine clinical practice, a change of culture and mindset is needed. Only focusing on patient directed interventions will not suffice to accomplish this. Finally, public awareness for the right to engage in SDM should be raised. Patients should know where to find relevant information regarding their medical condition and available options at all times. More patient directed interventions such as PtDAs should be made available, either by developing new interventions or by translating and adapting PtDAs from the international scene. By implementing these recommendations, we could truly start our journey towards patient-centered care.status: accepte