Newly synthesized palladium(II) complexes with dialkyl esters of (S,S)-propylenediamine-N,N'-di-(2,2'-di-(4-hydroxy-benzil))acetic acid: in vitro investigation of biological activities and HSA/DNA binding.

The four new ligands, dialkyl esters of (S,S)-propylenediamine-N,N'-di-(2,2'-di-(4-hydroxy-benzil))acetic acid (R2-S,S-pddtyr·2HCl) (R = ethyl (L1), propyl (L2), butyl (L3), and pentyl (L4)) and corresponding palladium(II) complexes have been synthesized and characterized by microanalysis, infrared, 1H NMR and 13C NMR spectroscopy. In vitro cytotoxicity was evaluated using the MTT assay on four tumor cell lines, including mouse mammary (4T1) and colon (CT26), and human mammary (MDA-MD-468) and colon (HCT116), as well as non-tumor mouse mesenchymal stem cells. Using fluorescence spectroscopy were investigated the interactions of new palladium(II) complexes [PdCl2(R2-S,S-pddtyr)]; (R = ethyl (C1), propyl (C2), butyl (C3), and pentyl (C4)) with calf thymus human serum albumin (HSA) and DNA (CT-DNA). The high values of the binding constants, Kb, and the Stern-Volmer quenching constant, KSV, show the good binding of all complexes for HSA and CT-DNA. The mentioned ligands and complexes were also tested on in vitro antimicrobial activity against 11 microorganisms. Testing was performed by the microdilution method, where the minimum inhibitory concentration (MMC) and the minimum microbicidal concentration (MMC) were determined.

Synthesis, characterization, HSA binding, molecular docking, cytotoxicity study, and antimicrobial activity of new palladium(II) complexes with propylenediamine derivatives of phenylalanine.

The four new ligands, propylenediamine derivatives of phenylalanine (R2-S,S-pddbaˑ2HCl; L1-L4) and their palladium(II) complexes (C1-C4) were synthesized and characterized by elemental analysis, infrared, 1H and 13C NMR spectroscopy. The interactions of new palladium(II) complexes with human serum albumin (HSA) were studied by fluorescence spectroscopy. All investigated compounds can be transported to target cells by binding to HSA, but complex C4 interacts most strongly. Molecular docking simulations were applied to comprehend the binding of the complex to the molecular target of HSA. Obtained results are in good correlations with experimental data regarding binding affinity by HSA. In vitro cytotoxicity activities were investigated on four tumor cell lines (mouse mammary (4 T1) and colon (CT26), human mammary (MDA-MD-468) and colon (HCT116)) and mouse mesenchymal stem cells as non-tumor control cells. Cytotoxic capacity was determined by MTT test and according to obtained results ligand L4 stands out as the most active and selective compound and as a good candidate for future in vivo testing. Further examination of the ligand L4 and corresponding complex C4 led to the conclusion that both induced cell death mainly by apoptosis. Ligand L4 facilitated cycle arrest in G0/G1 phase and decreased proliferative capacity of tumor cells. In vitro antimicrobial activity for ligands and corresponding Pd(II) complexes was investigated against eleven microorganisms (eight strains of pathogenic bacteria and three yeast species) using microdilution method. The minimum inhibitory concentration and minimum microbicidal concentration were determined.

Primary antiphospholipid syndrome as a cause of impaired left ventricular diastolic function: experience from a Serbian cohort.

OBJECTIVES: Cardiovascular manifestations, encountered in antiphospholipid syndrome, may develop as a consequence of acquired thrombophilia mediated by antiphospholipid antibodies and accelerated atherosclerosis as well. Our study aims to assess the impairment of the left ventricular diastolic performance, as early evidence of myocardial involvement in primary antiphospholipid syndrome (PAPS). METHODS: We analysed 101 PAPS patients, with the average age of 47.70±13.14y. Anticardiolipin antibodies (aCL IgG/IgM), anti-ß2 glycoprotein-I (anti-ß2GPI IgG/IgM), and lupus anticoagulant (LAC) were determined. Abnormal cut-off values used for left ventricular diastolic dysfunction (LVDD) were septal E ́<7 cm/sec, lateral E ́ <10 cm/sec, average E/E ́ ratio >14, LA volume index (LAVI) >34 mL/m2, and peak tricuspid regurgitation velocity >2.8 m/sec. LVDD was present if more than half parameters were with abnormal values. The results were compared to 90 healthy, age and sex-matched controls. RESULTS: LVDD was significantly more prevalent in PAPS patients compared to healthy controls (24.8% vs. 2.2%, p=0.001). In PAPS patients, it was signi cantly related to age, body mass index, hyperlipidaemia, thromboses and LAC positivity (p=0.0001, p=0.008, p=0.039, p=0.001, p=0.047 respectively). Patients with PAPS had higher LAVI (29.76±6.40 ml/m2 vs. 26.62±7.8 ml/m2, p=0.012), higher isovolumic relaxation time, lower lateral É velocity and lower E/É ratio compared to controls (p=0.0001, p=0.020, p=0.038, respectively). In multivariate analysis, thromboses in PAPS were significant, and independent predictors of LVDD. CONCLUSIONS: Thrombotic PAPS patients are at higher risk of LVDD development. Strong action against standard atherosclerotic risk factors and adequate therapy regimes seems to be crucial to preserve good diastolic performance of the left ventricle in PAPS.

Synthesis, characterization, DNA interactions and biological activity of new palladium(II) complexes with some derivatives of 2-aminothiazoles.

Newly palladium(II) complexes (C1, C2) with derivatives of 2-aminothiazoles (L1 = 2-amino-6-methylbenzothiazole, L2 = 2-amino-6-chlorobenzothiazole), general formula [PdL2Cl2] were synthesized and characterized by elemental microanalyses, IR, NMR spectroscopy and X-ray spectroscopy in case of [Pd(L2)2Cl2]. The kinetic of the substitution reactions of complexes and the nucleophiles, such as guanosine-5'-monophosphate (5'-GMP), tripeptide glutathione (GSH) and amino acid L-methionine (L-Met), were studied by stopped-flow technique. The complex C2 was always more reactive, while the order of the reactivity of the nucleophiles, due to the associative mode of the reaction, was L-Met > GSH > 5'-GMP. In order to determine the type of interactions between palladium(II) complexes and calf thymus DNA (CT-DNA), we used electronic absorption spectroscopy, viscosity measurements, and fluorescence spectroscopic studies, while interactions with bovine serum albumin (BSA) were determined only with fluorescence spectroscopic studies. The observed results confirmed that both complexes bound to DNA by groove binding. The significantly strong interaction with BSA, especially for complex C2, was also observed. In vitro cytotoxic activity was evaluated against four tumor cell lines, 4 T1, CT26, MDA-MB-468, HCT116 and mesenchymal stem cells (mMSC). C1 complex showed higher cytotoxic activity against CT26 cell line. Flow cytometry analysis showed that C1 stimulated apoptosis of tumor cells via inhibition of expression of antiapoptotic Bcl-2 molecule and decelerated proliferation by decreasing Cyclin-D and increasing expression of P21. In vitro antimicrobial activity for ligands and corresponding palladium(II) complexes was investigated by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. Tested compounds exhibited selective and moderate activity.

The Accuracy of Serum Biomarkers in the Diagnosis of Steatosis, Fibrosis, and Inflammation in Patients with Nonalcoholic Fatty Liver Disease in Comparison to a Liver Biopsy.

Background and Objective: This study was conducted to evaluate the diagnostic performance of various biomarkers for steatosis, fibrosis, and inflammation in comparison to a liver biopsy (LB) in patients with nonalcoholic fatty liver disease (NAFLD). Materials and Methods: This was a cross-sectional study that included 135 patients with biopsy-proven NAFLD. Fatty liver index (FLI), hepatic steatosis index (HSI), cell death markers (CK-18 M30 and CK-18 M65), FIB-4 index, NAFLD fibrosis score (NFS), BARD, and AST to platelet ratio index (APRI) were calculated and analysed. Results: FLI, HSI scores, and the cell death biomarkers showed poor diagnostic accuracy for steatosis detection and quantification, with an area under the curve (AUC) of <0.70. The cell death biomarkers likewise did not perform well for the detection of nonalcoholic steatohepatitis (NASH) (AUC < 0.7). As for the fibrosis staging, only APRI and the cell death biomarkers had moderate accuracy (AUC > 0.7) for advanced fibrosis, whereas FIB-4, BARD, and NFS scores demonstrated poor performance (AUC < 0.70). However, a combination of FIB-4 and NFS with the cell death biomarkers had moderate accuracy for advanced (≥F3) fibrosis detection, with an AUC of >0.70. Conclusions: In this first study on Croatian patients with NAFLD, serum biomarkers demonstrated poor diagnostic performance for the noninvasive diagnosis of liver steatosis and NASH. APRI and the cell death biomarkers had only moderate accuracy for diagnosing advanced fibrosis, as did the combination of FIB-4 and NFS with the cell death biomarkers. Further studies regarding serum biomarkers for all NAFLD stages are needed.

Dimerisation of the Yeast K+ Translocation Protein Trk1 Depends on the K+ Concentration.

In baker's yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K+ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K+ concentration.

Newly synthesized palladium(II) complexes with aminothiazole derivatives: in vitro study of antimicrobial activity and antitumor activity on the human prostate cancer cell line.

Five new complexes of the palladium(II) ion (C1-C5) having the general formula [(PdL2)]Cl2 with some 2-aminothiazoles (L1-L5), where L1 = 2-amino-4-(3,4-difluorophenyl)thiazole, L2 = 2-amino-5-methyl-4-phenylthiazole, L3 = 2-amino-4-phenylthiazole, L4 = 2-amino-4-(4-chlorophenyl)thiazole, and L5 = 2-amino-4-(2,4-difluorophenyl)thiazole, have been synthesized and characterized by elemental microanalysis and infrared, 1H NMR and 13C NMR spectroscopy. The in vitro antimicrobial activity of the five ligands and the corresponding Pd(II) complexes is investigated. Testing is performed by the microdilution method and the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) have been determined. Testing is conducted against 11 microorganisms (nine strains of pathogenic bacteria and two yeast species). The tested ligands and palladium(II) complexes show selective, high and moderate activity. There is a difference in antimicrobial activity between the ligands and the corresponding palladium(II) complexes. The complexes have significant anti-staphylococcal activity and activity on Pseudomonas aeruginosa which is better than the positive control. The interactions of newly synthesized palladium(II) complexes with calf thymus DNA (CT-DNA) were investigated using UV-Vis absorption and fluorescence spectroscopy. Analysis of UV-absorption and fluorescence spectra indicates the formation of a complex between the palladium(II) complexes and DNA. The high values of intrinsic binding constants, Kb, of the order 104 M-1 and Stern-Volmer quenching constants, KSV, of the order 105 M-1 indicated very good binding of all complexes to CT-DNA. Also, the new Pd(II) complexes show high cytotoxic activity towards the human prostate cancer cell line and insignificant activity towards non-cancerous human fibroblasts. Future research could additionally explore the biological activity of Pd(II) complexes presented in this paper and investigate the possibility of their implementation in clinical practice.

Liver disease in the era of COVID-19: Is the worst yet to come?

The global social, economic and political crises related to coronavirus disease 2019 (COVID-19) presumably had more indirect than direct negative impacts on health systems. Drastic lifestyle changes, social isolation and distancing, and individual and global financial crises resulted in robust populations forfeiting healthy habits and seeking comfort in alcoholic beverages, drugs and unhealthy diets. The inevitable consequences are increases in the incidence of nonalcoholic fatty liver disease, viral hepatitis, acute alcoholic hepatitis, liver cirrhosis decompensation and ultimately liver-related mortality. The inaccessibility of regular clinical and sonographic monitoring systems has caused difficulties in the treatment of patients with chronic liver disease (CLD) and has prevented prompt hepatocellular carcinoma detection and treatment. A dramatic reduction in the number of liver donors and the transformation of numerous transplantation centers into COVID-19 units drastically decreased the rate of orthotopic liver transplantation. The indirect, unavoidable effects of the COVID-19 pandemic in the following years have yet to be determined. Substantial efforts in the management of patients with liver disease in order to overcome the inevitable COVID-19-related morbidity and mortality that will follow have yet to be initiated. Several questions regarding the impact of the COVID-19 pandemic on liver disease remain. The most important question for general CLD patients is: How will the modification of clinical practice during this pandemic affect the outcomes of CLD patients? This article reviews the influence of COVID-19 on patients with liver disease during the pandemic, with particular emphasis on the disease course associated with pandemic resolution.

Non-Alcoholic Fatty Liver Disease and COVID-19-Two Pandemics Hitting at the Same Time.

The COVID-19 pandemic was and still is a global burden with more than 178,000,000 cases reported so far. Although it mainly affects respiratory organs, COVID-19 has many extrapulmonary manifestations, including, among other things, liver injury. Many hypotheses have been proposed to explain direct and indirect impacts of the SARS-CoV-2 virus on the liver. Studies have shown that around 15-30% of patients with COVID-19 have underlying liver disease, and 20-35% of patients with COVID-19 had altered liver enzymes at admission. One of the hypotheses is reactivation of an underlying liver disease, such as non-alcoholic fatty liver disease (NAFLD). Some studies have shown that NAFLD is associated with severe COVID-19 and poor outcome; nevertheless, other studies showed no significant difference between groups in comparing complications and clinical outcomes. Patients with NAFLD may suffer severe COVID-19 due to other comorbidities, especially cardiovascular diseases. The link between NAFLD and COVID-19 is not clear yet, and further studies and research are needed.

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.

Accuracy of Controlled Attenuation Parameter and Liver Stiffness Measurement in Patients with Non-alcoholic Fatty Liver Disease.

We evaluated the diagnostic accuracy of the controlled attenuation parameter (CAP) and liver stiffness measurements (LSM) measured with either an M or XL probe against liver biopsy (LB) in patients with non-alcoholic fatty liver disease (NAFLD). This study was a cross-sectional prospective study that included 179 NAFLD patients. With a cutoff value for CAP ≥345, we can exclude significant steatosis in 87% (79.4%-92.5%) of our population. With respect to the LSM, the highest accuracy was obtained for F ≥ F3 (area under the receiver operating characteristic curve [AUROC] = 0.98) and F = F4 (AUROC = 0.98). In a multivariable linear regression model, significant predictors influencing LSM were fibrosis stage (ß = 2.6, p < 0.001) as a positive predictor and lobular inflammation (ß = -0.68, p = 0.04) as a negative predictor, without significant influence after adjustment for CAP and probe type. We found that CAP is a satisfactory method for excluding advanced steatosis, while LSM is a good non-invasive marker for the exclusion of fibrosis.

Pre-existing liver disease in pregnancy: Cirrhosis, autoimmune hepatitis and liver transplantation.

Liver disease during pregnancy is one of the least studied topics, but it links the interests of hepatologists, gynaecologists and family physicians. Approximately 3% of pregnant woman experience some type of liver disease. Liver disease can occur as a result of pregnancy, before pregnancy and coincidence-related during pregnancy. Pregnancy in women with pre-existing liver disease is essential that the clinicians are familiar with this disorder so they can respond promptly and appropriately in all of these situations. So, because of the complications for both mother and child, it is important that liver disease is recognized in a timely manner to avoid undesirable outcomes.

Vitamin D Deficiency: Consequence or Cause of Obesity?

Obesity is defined as an excess amount of body fat and represents a significant health problem worldwide. High prevalence of vitamin D (VD) deficiency in obese subjects is a well-documented finding, most probably due to volumetric dilution into the greater volumes of fat, serum, liver, and muscle, even though other mechanisms could not completely be excluded, as they may contribute concurrently. Low VD could not yet be excluded as a cause of obesity, due to its still incompletely explored effects through VD receptors found in adipose tissue (AT). VD deficiency in obese people does not seem to have consequences for bone tissue, but may affect other organs, even though studies have shown inconsistent results and VD supplementation has not yet been clearly shown to benefit the dysmetabolic state. Hence, more studies are needed to determine the actual role of VD deficiency in development of those disorders. Thus, targeting lifestyle through healthy diet and exercise should be the first treatment option that will affect both obesity-related dysmetabolic state and vitamin D deficiency, killing two birds with one stone. However, VD supplementation remains a treatment option in individuals with residual VD deficiency after weight loss.

Prevention of Infectious Complications in Acute Pancreatitis: Results of a Single-Center, Randomized, Controlled Trial.

OBJECTIVES: This study aimed to investigate the efficiency of imipenem to prevent infectious complications in predicted severe acute pancreatitis (AP). METHODS: Consecutive AP patients were randomized to imipenem 3 × 500 mg intravenously daily or an identical placebo. Exclusion criteria were prior AP, chronic pancreatitis, active malignancy, immune deficiency, active infection, concomitant antibiotic treatment, pregnancy, and patients younger than 18 years. Infectious complications including infected pancreatic necrosis, pneumonia, urinary tract infection, positive blood cultures, sepsis, and other infections were assessed as the primary outcome. Secondary outcomes included mortality, persistent organ failure, systemic inflammatory response syndrome, local complications, serious adverse events, and need for surgical intervention. RESULTS: Forty-nine patients were randomized to each group. Infectious complications were present in 10 versus 12 of 49 patients (relative risk [RR], 0.833; 95% confidence interval [CI], 0.398-1.747). There were no significant differences in infected pancreatic necrosis (RR, 1.5; 95% CI, 0.262-8.588), pneumonia (RR, 1.5; 95% CI, 0.262-8.588), urinary tract infection (RR, 0.6; 95% CI, 0.152-2.374), positive blood cultures (RR, 0.5; 95% CI, 0.047-5.336), sepsis (RR, 0.333; 95% CI, 0.036-3.095), and other (RR, 1.333; 95% CI, 0.315-5.648). We found no significant differences in secondary outcomes. CONCLUSIONS: Concordantly to available evidence, there is currently no ground to support prophylactic use of antibiotics in predicted severe AP.

Epidemiological and clinical features of primary biliary cholangitis in two Croatian regions: a retrospective study.

AIM: To assess the measures of disease frequency and determine the clinical features of primary biliary cholangitis (PBC) in two Croatian regions. METHODS: Databases of two tertiary hospitals, one located in the continental and one in the coastal region of Croatia, were retrospectively searched for PBC patients diagnosed from 2007 to 2018. Epidemiologic data analysis was restricted to patients from each hospital's catchment area. We analyzed factors related to response to therapy and event-free survival (EFS), defined as absence of ascites, variceal bleeding, encephalopathy, hepatocellular carcinoma, liver transplantation (LT), or death. In addition, we determined clinical and demographic data of transplanted PBC patients. RESULTS: Out of 83 PBC patients, 86.7% were female, with a median age at diagnosis of 55 years. Average PBC incidence for the 11-year period was 0.79 and 0.89 per 100000 population, whereas the point prevalence on December 31, 2017 was 11.5 and 12.5 in the continental and coastal region, respectively. Of 76 patients with complete medical records, 21% had an advanced disease stage, 31.6% had an associated autoimmune condition, and all received ursodeoxycholic acid. EFS rate at 5 years was 95.8%. In an age and sex-adjusted multivariate Cox regression model, the only factor significantly associated with inferior EFS was no response to therapy (HR=18.4; P=0.018). Of all Croatian patients who underwent LT, 3.8% had PBC, with the survival rate at 5 years after LT of 93.4%. CONCLUSION: This study gives pioneer insights into the epidemiological and clinical data on PBC in Croatia, thus complementing the PBC map of Southeast Europe.

Advances in diagnosis of chronic liver diseases in pediatric patients.

BACKGROUND: Chronic liver diseases (CLD) present important clinical problem in children with various age-dependent causes. Nonalcoholic fatty liver disease (NAFLD) with its increasing prevalence is a major problem with regard to its timely recognition and the need for long-term disease monitoring. At present, a perfect non-invasive method for the evaluation of liver fibrosis is not available. METHODS: A non-systematic literature search was performed to summarize the current knowledge about transient elastography (TE) with controlled attenuation parameter (CAP) in children with CLD. Ovid MEDLINE, Ovid EMBASE, Google scholar, and The Cochrane Library databases were searched for relevant articles evaluating TE in the pediatric population. RESULTS: Normal values of liver stiffness measurements (LSM) according to the age are given, as well as the advantages and disadvantages of the method. The utility of TE in specific liver disease in pediatric population is summarized. CONCLUSIONS: TE with CAP is a valuable non-invasive method for the liver-damage assessment. Clinical interpretation of TE results should be made in parallel with the assessment of the patient's demographics, disease etiology, and essential laboratory parameters.

Liver Disease During Pregnancy: A Challenging Clinical Issue.

One of the least studied topics in the field of obstetrics is liver disease during pregnancy, which creates a challenge for both gynecologists and hepatologists. Approximately 3% of pregnant women are affected by some form of liver disease during pregnancy. Some of these conditions can be fatal for both the mother and child. In addition, 3 types of liver disease need to be differentiated during pregnancy. One type is liver disease directly related to pregnancy, which can occur at a specific time during pregnancy. Another type is liver disease not related to pregnancy, which can occur at any time, such as viral- or drug-induced hepatitis. Furthermore, pregnancy can occur in women with pre-existing liver disease. It is essential that the clinicians are familiar with this disorder so they can respond promptly and appropriately in all of these situations, especially when emergency delivery is needed and must not be postponed.

Nonalcoholic fatty liver disease and liver transplantation - Where do we stand?

Nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) is a challenging and multisystem disease that has a high socioeconomic impact. NAFLD/NASH is a main cause of macrovesicular steatosis and has multiple impacts on liver transplantation (LT), on patients on the waiting list for transplant, on post-transplant setting as well as on organ donors. Current data indicate new trends in the area of chronic liver disease. Due to the increased incidence of metabolic syndrome (MetS) and its components, NASH cirrhosis and hepatocellular carcinoma caused by NASH will soon become a major indication for LT. Furthermore, due to an increasing incidence of MetS and, consequently, NAFLD, there will be more steatotic donor livers and less high quality organs available for LT, in addition to a lack of available liver allografts. Patients who have NASH and are candidates for LT have multiple comorbidities and are unique LT candidates. Finally, we discuss long-term grafts and patient survival after LT, the recurrence of NASH and NASH appearing de novo after transplantation. In addition, we suggest topics and areas that require more research for improving the health care of this increasing patient population.