Croatian guidelines for the diagnosis and treatment of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a term describing excessive accumulation of fat in hepatocytes, and is associated with metabolic syndrome and insulin resistance. NAFLD prevalence is on increase and goes in parallel with the increasing prevalence of metabolic syndrome and its components. That is why Croatian guidelines have been developed, which cover the screening protocol for patients with NAFLD risk factors, and the recommended diagnostic work-up and treatment of NAFLD patients. NAFLD screening should be done in patients with type 2 diabetes mellitus, or persons with two or more risk factors as part of metabolic screening, and is carried out by noninvasive laboratory and imaging methods used to detect fibrosis. Patient work-up should exclude the existence of other causes of liver injury and determine the stage of fibrosis as the most important factor in disease prognosis. Patients with initial stages of fibrosis continue to be monitored at the primary healthcare level with the management of metabolic risk factors, dietary measures, and increased physical activity. Patients with advanced fibrosis should be referred to a gastroenterologist/ hepatologist for further treatment, monitoring, and detection and management of complications

Molecular dynamics studies of the steps in the catalytic cycle of gamma-secretase: searching for novel drug-design strategies for Alzheimer's disease

Background: Alzheimer’s disease is the most common type of dementia, and currently the biggest financial burden for health care providers in developed countries. γ-secretase is a large membrane embedded protease and a major target in drug development efforts based on over 250 mutations in its structure that can cause the disease. We used all-atom and coarse-grained molecular dynamic studies to describe the structural changes in the catalytic cycle of γ-secretase. Results: Adaptive Poisson-Boltzmann calculations showed that γ-secretase is a highly polarized enzyme that can attract N-terminal and C-terminal parts of its protein substrate. The presenilin subunit of γ-secretase has a cluster of positive residues that can stabilize the negatively charged C-terminal of the catalytic intermediates. The nicastrin subunit can close above the catalytic site like a lid or rotate above the enzyme. The 6 Å distance between the active site aspartates showed that the enzyme structure from the Protein Data Bank represents an inactive enzyme. In silico mutations showed that 4.5 Å distance between the active site aspartates is optimal for catalysis. Asparagines in place of the active site aspartates can be used to mimic the active enzyme in structural studies of substrate binding steps. The cholesterol-rich membrane supports the optimal distance between the active site aspartates, what is consistent with experimental results which showed that cholesterol is crucial for the enzyme activity. Disease causing mutation G384A can increase the distance between the active site aspartates, what can explain experimentally observed pathogenic decrease in the activity of mutated enzyme. Conclusion: Molecular dynamic studies can be used for the development of drug candidates that can specifically target changes in γ-secretase structure that support the pathogenic changes in the enzyme activity.Pozadina: Alzheimerova bolest je najčešći oblik demencije i trenutno najveći financijski teret za zdravstvene sustave u razvijenim zemljama. γ-sekretaza je velika membranska proteaza koja je postala važna meta u pokušajima razvoja lijekova zbog preko 250 mutacija u njenoj strukturi, koje mogu izazvati bolest. U ovom radu, pomoću coarse-grained i all-atom studija molekularne dinamike opisali smo strukturne promjene u katalitičkom ciklusu γ-sekretaze. Rezultati: Adaptivni Poisson-Boltzmann izračuni pokazali su da je γ-sekretaza vrlo polariziran enzim koji može privući N-terminalni i C-terminalni dio svog supstrata. Presenilinska podjedinica γ-sekretaze, sadrži klaster pozitivnih aminokiselina koje mogu stabilizirati negativno nabijeni C-terminalni dio katalitičkih međuprodukata. Nikastrinska podjedinica se može poput poklopca preklopiti iznad katalitičkog mjesta te rotirati iznad enzima. Udaljenost od 6 Å između aspartata u aktivnom mjestu pokazala je da struktura enzima dostupna na Protein Data Bank predstavlja inaktivni enzim. In silico mutacije pokazale su da je udaljenost od 4.5 Å između aspartata optimalna za katalizu. Asparagini umjesto aspartata u aktivnom mjestu mogu služiti za oponašanje aktivnog enzima kod istraživanja strukture u koracima vezanja supstrata. Optimalna udaljenost između aspartata u aktivnom mjestu je u membranama bogatim kolesterolom, što se slaže sa rezultatima eksperimenata koji su pokazali da je kolesterol presudan za enzimsku aktivnost. G384A mutacija, koja izaziva bolest, može povećati udaljenost između aspartata u aktivnom mjestu, što može objasniti eksperimentalno potvrđeno patogeno smanjenje aktivnosti mutiranog enzima. Zaključak: Studije molekularne dinamike se mogu koristiti za razvoj potencijalnih lijekova koji će specifično ciljati na promjene u strukturi γ-sekretaze koje vode do patogenih promjena u enzimskoj aktivnosti

Molecular dynamics studies of the steps in the catalytic cycle of gamma-secretase: searching for novel drug-design strategies for Alzheimer's disease

Background: Alzheimer’s disease is the most common type of dementia, and currently the biggest financial burden for health care providers in developed countries. γ-secretase is a large membrane embedded protease and a major target in drug development efforts based on over 250 mutations in its structure that can cause the disease. We used all-atom and coarse-grained molecular dynamic studies to describe the structural changes in the catalytic cycle of γ-secretase. Results: Adaptive Poisson-Boltzmann calculations showed that γ-secretase is a highly polarized enzyme that can attract N-terminal and C-terminal parts of its protein substrate. The presenilin subunit of γ-secretase has a cluster of positive residues that can stabilize the negatively charged C-terminal of the catalytic intermediates. The nicastrin subunit can close above the catalytic site like a lid or rotate above the enzyme. The 6 Å distance between the active site aspartates showed that the enzyme structure from the Protein Data Bank represents an inactive enzyme. In silico mutations showed that 4.5 Å distance between the active site aspartates is optimal for catalysis. Asparagines in place of the active site aspartates can be used to mimic the active enzyme in structural studies of substrate binding steps. The cholesterol-rich membrane supports the optimal distance between the active site aspartates, what is consistent with experimental results which showed that cholesterol is crucial for the enzyme activity. Disease causing mutation G384A can increase the distance between the active site aspartates, what can explain experimentally observed pathogenic decrease in the activity of mutated enzyme. Conclusion: Molecular dynamic studies can be used for the development of drug candidates that can specifically target changes in γ-secretase structure that support the pathogenic changes in the enzyme activity.Pozadina: Alzheimerova bolest je najčešći oblik demencije i trenutno najveći financijski teret za zdravstvene sustave u razvijenim zemljama. γ-sekretaza je velika membranska proteaza koja je postala važna meta u pokušajima razvoja lijekova zbog preko 250 mutacija u njenoj strukturi, koje mogu izazvati bolest. U ovom radu, pomoću coarse-grained i all-atom studija molekularne dinamike opisali smo strukturne promjene u katalitičkom ciklusu γ-sekretaze. Rezultati: Adaptivni Poisson-Boltzmann izračuni pokazali su da je γ-sekretaza vrlo polariziran enzim koji može privući N-terminalni i C-terminalni dio svog supstrata. Presenilinska podjedinica γ-sekretaze, sadrži klaster pozitivnih aminokiselina koje mogu stabilizirati negativno nabijeni C-terminalni dio katalitičkih međuprodukata. Nikastrinska podjedinica se može poput poklopca preklopiti iznad katalitičkog mjesta te rotirati iznad enzima. Udaljenost od 6 Å između aspartata u aktivnom mjestu pokazala je da struktura enzima dostupna na Protein Data Bank predstavlja inaktivni enzim. In silico mutacije pokazale su da je udaljenost od 4.5 Å između aspartata optimalna za katalizu. Asparagini umjesto aspartata u aktivnom mjestu mogu služiti za oponašanje aktivnog enzima kod istraživanja strukture u koracima vezanja supstrata. Optimalna udaljenost između aspartata u aktivnom mjestu je u membranama bogatim kolesterolom, što se slaže sa rezultatima eksperimenata koji su pokazali da je kolesterol presudan za enzimsku aktivnost. G384A mutacija, koja izaziva bolest, može povećati udaljenost između aspartata u aktivnom mjestu, što može objasniti eksperimentalno potvrđeno patogeno smanjenje aktivnosti mutiranog enzima. Zaključak: Studije molekularne dinamike se mogu koristiti za razvoj potencijalnih lijekova koji će specifično ciljati na promjene u strukturi γ-sekretaze koje vode do patogenih promjena u enzimskoj aktivnosti

The Binding of Different Substrate Molecules at the Docking Site and the Active Site of γ-Secretase Can Trigger Toxic Events in Sporadic and Familial Alzheimer’s Disease

Pathogenic changes in γ-secretase activity, along with its response to different drugs, can be affected by changes in the saturation of γ-secretase with its substrate. We analyze the saturation of γ-secretase with its substrate using multiscale molecular dynamics studies. We found that an increase in the saturation of γ-secretase with its substrate could result in the parallel binding of different substrate molecules at the docking site and the active site. The C-terminal domain of the substrate bound at the docking site can interact with the most dynamic presenilin sites at the cytosolic end of the active site tunnel. Such interactions can inhibit the ongoing catalytic activity and increase the production of the longer, more hydrophobic, and more toxic Aβ proteins. Similar disruptions in dynamic presenilin structures can be observed with different drugs and disease-causing mutations. Both, C99-βCTF-APP substrate and its different Aβ products, can support the toxic aggregation. The aggregation depends on the substrate N-terminal domain. Thus, the C99-βCTF-APP substrate and β-secretase path can be more toxic than the C83-αCTF-APP substrate and α-secretase path. Nicastrin can control the toxic aggregation in the closed conformation. The binding of the C99-βCTF-APP substrate to γ-secretase can be controlled by substrate channeling between the nicastrin and β-secretase. We conclude that the presented two-substrate mechanism could explain the pathogenic changes in γ-secretase activity and Aβ metabolism in different sporadic and familial cases of Alzheimer’s disease. Future drug-development efforts should target different cellular mechanisms that regulate the optimal balance between γ-secretase activity and amyloid metabolism

The Binding of Different Substrate Molecules at the Docking Site and the Active Site of γ-Secretase Can Trigger Toxic Events in Sporadic and Familial Alzheimer’s Disease

Pathogenic changes in γ-secretase activity, along with its response to different drugs, can be affected by changes in the saturation of γ-secretase with its substrate. We analyze the saturation of γ-secretase with its substrate using multiscale molecular dynamics studies. We found that an increase in the saturation of γ-secretase with its substrate could result in the parallel binding of different substrate molecules at the docking site and the active site. The C-terminal domain of the substrate bound at the docking site can interact with the most dynamic presenilin sites at the cytosolic end of the active site tunnel. Such interactions can inhibit the ongoing catalytic activity and increase the production of the longer, more hydrophobic, and more toxic Aβ proteins. Similar disruptions in dynamic presenilin structures can be observed with different drugs and disease-causing mutations. Both, C99-βCTF-APP substrate and its different Aβ products, can support the toxic aggregation. The aggregation depends on the substrate N-terminal domain. Thus, the C99-βCTF-APP substrate and β-secretase path can be more toxic than the C83-αCTF-APP substrate and α-secretase path. Nicastrin can control the toxic aggregation in the closed conformation. The binding of the C99-βCTF-APP substrate to γ-secretase can be controlled by substrate channeling between the nicastrin and β-secretase. We conclude that the presented two-substrate mechanism could explain the pathogenic changes in γ-secretase activity and Aβ metabolism in different sporadic and familial cases of Alzheimer’s disease. Future drug-development efforts should target different cellular mechanisms that regulate the optimal balance between γ-secretase activity and amyloid metabolism

The Binding of Different Substrate Molecules at the Docking Site and the Active Site of γ-Secretase Can Trigger Toxic Events in Sporadic and Familial Alzheimer’s Disease

Pathogenic changes in γ-secretase activity, along with its response to different drugs, can be affected by changes in the saturation of γ-secretase with its substrate. We analyze the saturation of γ-secretase with its substrate using multiscale molecular dynamics studies. We found that an increase in the saturation of γ-secretase with its substrate could result in the parallel binding of different substrate molecules at the docking site and the active site. The C-terminal domain of the substrate bound at the docking site can interact with the most dynamic presenilin sites at the cytosolic end of the active site tunnel. Such interactions can inhibit the ongoing catalytic activity and increase the production of the longer, more hydrophobic, and more toxic Aβ proteins. Similar disruptions in dynamic presenilin structures can be observed with different drugs and disease-causing mutations. Both, C99-βCTF-APP substrate and its different Aβ products, can support the toxic aggregation. The aggregation depends on the substrate N-terminal domain. Thus, the C99-βCTF-APP substrate and β-secretase path can be more toxic than the C83-αCTF-APP substrate and α-secretase path. Nicastrin can control the toxic aggregation in the closed conformation. The binding of the C99-βCTF-APP substrate to γ-secretase can be controlled by substrate channeling between the nicastrin and β-secretase. We conclude that the presented two-substrate mechanism could explain the pathogenic changes in γ-secretase activity and Aβ metabolism in different sporadic and familial cases of Alzheimer’s disease. Future drug-development efforts should target different cellular mechanisms that regulate the optimal balance between γ-secretase activity and amyloid metabolism

Role of motivation in serving a suspended sentence with protective supervision: perspectives of offenders and probation officers

Tijekom izvršavanja uvjetne osude sa zaštitnim nadzorom probacijski službenici imaju dvostruku profesionalnu ulogu s jedinstvenom svrhom odvraćanja osuđenika od daljnjeg kriminalnog ponašanja i smanjivanja vjerojatnosti recidivizma. S jedne strane njihov zadatak je nadzirati slobodu osuđenika (počinitelja kaznenih djela) i njihovo pridržavanje obveza, dok s druge strane trebaju uspostaviti kvalitetan profesionalni odnos i psihosocijalnim postupcima pomoći im u resocijalizaciji i reintegraciji unutar društvene zajednice. Ciljevi istraživanja bili su zahvaćanjem perspektive osuđenika i probacijskih službenika istražiti motivaciju osuđenika za izvršavanje uvjetne osude sa zaštitnim nadzorom kao jedne od probacijskih sankcija u Hrvatskoj, prije svega u kontekstu motivacije za promjenom ponašanja i pridržavanja uvjeta ove probacijske sankcije te steći uvid u eventualne promjene u motivaciji tijekom izvršavanja sankcije. Provedene su fokusne grupe s prigodnim uzorcima osuđenika (N=12) i probacijskih službenika (N=8). Rezultati su pokazali osuđenikovu tendenciju prema redovitom izvršavanju formalnih obveza uz postojanje pretežno ekstrinzične motivacije za promjenom te izostanak uviđanja svrhe sankcije, pogotovo na početku njezina izvršavanja. Tijekom izvršavanja sankcije dolazi do promjena u intenzitetu te djelomično i vrsti motivacije prije svega zbog uvida u dobrobit i korisnost sankcije čemu pridonosi kvaliteta odnosa s probacijskim službenikom i neki značajni životni događaji. Metode kojima službenici potiču osuđenike na izvršavanje sankcije i odvraćanje od budućih kaznenih djela usmjerene su usvajanju vještina rješavanja problema, osnaživanju socijalnog kapitala i osobne odgovornosti za odluke i vlastito ponašanje.When offenders serve a suspended sentence with protective supervision, their probation officers play a dual professional role with the sole purpose of deterring the offenders from further criminal behaviour and thereby reducing the likelihood of recidivism. Probation officers supervise offenders and monitor their compliance with different requirements, while at the same time establishing a high-quality professional relationship and conducting psychosocial procedures to assist offenders in re-socialisation and reintegration within the community. The aims of this research were to explore the perspectives of offenders and probation officers on the role of motivation when serving a suspended sentence with protective supervision, and to gain insights into possible changes in motivation during the serving of such a sentence. Focus groups were conducted with convenience samples of offenders (N=12) and probation officers (N=8). The results revealed that offenders, especially at the beginning of their sentence, showed predominantly extrinsic motivation when fulfilling the formal obligations of the sentence, without recognising their purpose. While serving the sentence, offenders’ motivation changed in intensity and partly in type, primarily because of increasing insight into the usefulness of the sentence, which was promoted by the quality of the relationship with the probation officer and some significant life events. In order to encourage offenders to comply with their sentence and refrain from future criminal behaviour, probation officers used problem-solving skills, combined with skills focused on empowering social capital and personal responsibility for decision-making and personal behaviour

Diagnostic performance of digital breast tomosynthesis in female patients with nipple discharge

Background: Nipple discharge is one of the most common symptoms related to the breast, but it is a presenting feature of breast cancer in 5%-12% of women. ----- Aims: The purpose of this study was to determine the diagnostic performance of digital breast tomosynthesis (DBT) in the evaluation of patients with nipple discharge and to compare it with mammography (MMG), ultrasound (US), and magnetic resonance imaging (MRI). ------ Methods and results: This retrospective study included 53 patients with nipple discharge. All patients underwent DBT, and results were compared to MMG, breast US, and MRI. Radiological findings for each method were categorized according to BI-RADS classification: categories 1-2 were considered negative and categories 3-5 positive. If a tissue specimen was obtained, the final diagnosis was established based on the results of histopathological analysis; otherwise, a clinical follow-up was required for at least 2 years to confirm benign radiological findings. Measures of diagnostic accuracy of DBT, MMG, US, and MRI were calculated and compared. ----- Results: Final histopathological analysis revealed six malignant breast lesions, all of which were detected in patients with pathologic nipple discharge. DBT and MRI exhibited high sensitivity (100%) and high negative predictive value (100%) for the detection of breast cancer in patients with nipple discharge. DBT showed higher specificity compared to MRI (82.9% vs. 61.9%). Sensitivity and specificity of MMG were 83.3% and 76.6%, respectively. Breast US was determined to have a sensitivity of 66.7% and specificity of 57.5%. ----- Conclusion: DBT exhibited higher specificity than MRI at the same level of sensitivity and negative predictive value. Therefore, the use of DBT should be considered as an alternative to MRI in the assessment of patients with nipple discharge

Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aβ42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aβ42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Aβ42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aβ42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research