Metabolic Associated Fatty Liver Disease (MAFLD) and COVID-19 Infection: An Independent Predictor of Poor Disease Outcome?

Background and Objectives: Early reports on COVID-19 infection suggested that the SARS-CoV-2 virus solely attacks respiratory tract cells. As the pandemic spread, it became clear that the infection is multiorganic. Metabolic associated fatty liver disease (MAFLD) is a chronic liver disease strongly associated with insulin resistance and diabetes. The aim of this study was to assess a possible interplay between MAFLD and COVID-19 infection and its implication in COVID-19 outcome. Materials and Methods: A retrospective observational study, including 130 COVID-19 positive patients was conducted. MAFLD diagnosis was made based on the International Consensus criteria. Patients were divided into two groups, group A (MAFLD) and group B (nonMAFLD). Anthropometric and laboratory analysis were obtained. COVID-19 severity was assessed using the NEWS2 score. Disease outcome was threefold and regarded as discharged, patients who required mechanical ventilation (MV), and deceased patients. Results: MAFLD prevalence was 42%, 67% of patients were discharged, and 19% needed MV. Mortality rate was 14%. MAFLD patients were significantly younger (p < 0.001), and had higher body mass index (p < 0.05), respiratory rate (p < 0.05) and systolic blood pressure (p < 0.05) than nonMAFLD patients. Regarding metabolic syndrome and inflammatory markers: group A had significantly higher glycemia at admission (p = 0.008), lower HDL-c (p < 0.01), higher triglycerides (p < 0.01), CRP (p < 0.001), IL-6 (p < 0.05) and ferritin (p < 0.05) than group B. MAFLD was associated with more prevalent type 2 diabetes (p = 0.035) and hypertension (p < 0.05). MAFLD patients had a more severe disease course (NEWS2 score, 6.5 ± 0.5 vs. 3 ± 1.0, p < 0.05). MAFLD presence was associated with lower patient discharge (p < 0.01) and increased need for MV (p = 0.024). Multiple regression analysis showed that BMI (p = 0.045), IL-6 (p = 0.03), and MAFLD (p < 0.05) are significant independent risk factors for a poor COVID-19 outcome. Conclusions: The prevalence of MAFLD is relatively high. MAFLD patients had a more severe COVID-19 clinical course and worse disease outcome. Our results imply that early patient stratification and risk assessment are mandatory in order to avoid poor outcomes.

Anxiety and Comorbidities Differences in Adults with Chronic Pulmonary Diseases: Serbian Single Center Experience.

Background and objectives: The purpose of this study is to investigate the differences in the degree of the anxiety and comorbidity levels in patients with different chronic pulmonary diseases such as chronic obstructive bronchitis (COPD) without emphysema phenotype, pulmonary emphysema, bronchial asthma and lung cancer. Materials and Methods: The prospective clinical study included 272 patients that were diagnosed and treated of pulmonary pathology. COPD (without emphysema phenotype) (Group-1), pulmonary emphysema (Group-2), bronchial asthma (Group-3) and lung cancer (Group-4) were assessed. For the evaluation of the anxiety degree, we used Hamilton Anxiety Rating Scale (HAM-A). Results: The degree of cardiovascular symptoms was significantly higher in Group-1 versus Group-2 (p < 0.001), Group-3 (p = 0.001) and Group-4 (p = 0.013), and significantly higher in Group-4 versus Group-2 (p = 0.046). The degree of respiratory symptoms was significantly higher in Group-1 versus Group-2 (p < 0.001), Group-3 (p < 0.001) and Group-4 (p = 0.002), and significantly higher in Group-4 versus Group-2 (p = 0.013) and versus Group-3 (p = 0.023). For gastrointestinal symptoms, the degree of one was significantly higher in Group-1 versus Group-2 (p < 0.001), Group-3 (p < 0.001) and Group-4 (p = 0.017). Somatic subscale values were significantly higher in Group-1 versus Group-2 (p < 0.001), Group-3 (p < 0.001) and Group-4 (p = 0.015), and significantly higher in Group-4 versus Group-2 (p = 0.024). Total HAM-A score was significantly higher in Group-1 versus Group-2 (p = 0.002) and Group-3 (p = 0.007). Conclusions: Patients with COPD (without emphysema phenotype) followed by the lung cancer are at elevated risk of being more mentally challenged in terms of increased anxiety. Furthermore, patients with exacerbation of evaluated pulmonary pathologies have various levels of comorbidities degrees.

The New Therapeutic Approaches in the Treatment of Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease which is characterized by extremely complex pathogenetic mechanisms and multifactorial etiology. Some of the many pathophysiological mechanisms involved in the development of NAFLD include oxidative stress, impaired mitochondrial metabolism, inflammation, gut microbiota, and interaction between the brain-liver-axis and the regulation of hepatic lipid metabolism. The new therapeutic approaches in the treatment of NAFLD are targeting some of these milestones along the pathophysiological pathway and include drugs like agonists of peroxisome proliferator-activated receptors (PPARs), glucagon-like peptide-1 (GLP-1) agonists, sodium/glucose transport protein 2 (SGLT2) inhibitors, farnesoid X receptor (FXR) agonists, probiotics, and symbiotics. Further efforts in biomedical sciences should focus on the investigation of the relationship between the microbiome, liver metabolism, and response to inflammation, systemic consequences of metabolic syndrome.

Impact of acute hyperglycemia on layer-specific left ventricular strain in asymptomatic diabetic patients: an analysis based on two-dimensional speckle tracking echocardiography.

BACKGROUND: Hyperglycemia has detrimental effect on ischemic myocardium, but the impact of acute hyperglycemia on the myocardium in asymptomatic diabetic patients has not been fully elucidated. Thus, this follow-up study was aimed to investigate the effects and reversibility of acute hyperglycemia on regional contractile function of left ventricle (LV) in diabetic patients without cardiovascular disease. METHODS: The two-dimensional speckle tracking echocardiography (2D-STE), including multilayer strain analysis, was used for evaluation of global and regional LV function in asymptomatic, normotensive patients with uncomplicated diabetes, with acute hyperglycemia ( ≥ 11.1 mmol/l) (Group A, n = 67), or with optimal metabolic control (fasting plasma glucose < 7 mmol/l and HbA1c < 7%) (Group B, n = 20), while 20 healthy individuals served as controls (Group C). In group A, after 72 h of i.v. continuous insulin treatment (at the time euglycemia was achieved) (second examination) and after 3 months following acute hyperglycemia (third examination) 2D-STE was repeated. RESULTS: Global longitudinal strain (GLS) (- 19.6 ± 0.4%) in Group A was significantly lower in comparison to both groups B (- 21.3 ± 0.4%; p < 0.05) and C (- 21.9 ± 0.4%; p < 0.01) at baseline, while we could not detect the differences between groups B and C. Peak systolic longitudinal endocardial (Endo), mid-myocardial (Mid) and epicardial (Epi) layer strain were significantly lower in group A at baseline compared to both groups B and C. Deterioration in peak systolic circumferential strain was observed at basal LV level, in all three layers (Endo, Mid and Epi) and in mid-cavity LV level in Epi layer in group A in comparison to group C. Moreover, in group A, after euglycemia was achieved (at second and third examination) GLS, as well as peak longitudinal and circumferential strain remain the same. CONCLUSION: Acute hyperglycemia in asymptomatic diabetic patients has significant negative effects on systolic LV myocardial mechanics primarily by reducing GLS and multilayer peak systolic longitudinal and circumferential strain which was not reversible after three months of good glycemic control.

The Inflammatory and Hemostatic Cardiovascular Risk Markers During Acute Hyperglycemic Crisis in Type 1 and Type 2 Diabetes.

BACKGROUND: We analyzed cardiovascular inflammatory (C-reactive protein (CRP), interleukin 6 (IL-6)), haemostatic (homocysteine) risk markers in lean and obese patients at admission and acute hyperglicemic crisis (AHC) resolving, involving diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS). METHODS: In that context, we included group A: N = 20 obese, B: N=20 lean patients with DKA; C: N = l0 obese, D: N=10 lean patients with HHS; E: N = 15 obese, F: N=15 lean controls. CRP IL-6, homocysteine were determined by ELISA. RESULTS: Our results showed that CRP IL-6, and homocysteine levels decreased in all groups: (A: p<0.001; B: p<0.001, C: p<0.05; D: p<0.001 mg/L), (A: p<0.001 B: p<0.001, C: p<0.001, D: p<0.01 pg/mL), (A: p<0.001, B: p <0.001; C: p<0.05, D: p=0.001 µmol/L), respectively, at resolving AHC. However, CRP persisted higher (p<0.001, p<0.01), IL-6 lower (p<0.05, p<0.001), while homocysteine levels turned out to be similar to controls. CONCLUSIONS: AHC is associated with increased inflammatory and hemostatic cardiovascular risk markers. Also, insulin therapy in AHC has had more pronounced favorable effect on IL-6 and homocystein than on CRP.

Polymorphisms of survivin -31 G/C gene are associated with risk of urothelial carcinoma in Serbian population.

PURPOSE: Survivin is thought to play an important role in carcinogenesis and is found to be associated with poor clinical outcome in various malignancies. Gene -31 G/C polymorphism has been identified as a risk factor for the development of several types of tumors. The purpose of this study was to investigate the association between survivin gene promoter -31C/G polymorphism and urothelial carcinoma (UC) risk in Serbian population and to compare the different expressions of survivin in UC of different disease stages, histological grades and tumor location in the upper or lower urinary tract. METHODS: DNA from 94 patients with primary UC and from 82 healthy subjects was subjected to PCR restriction fragment length polymorphism analysis (PCR-RFLP) to identify individual genotypes. UC samples were subjected to immunohistochemical analysis to assess survivin expression in these lesions. RESULTS: It was observed that the frequency of G/G genotype was greater in patients with UC (58.7%) than in controls (32%). Compared with study subjects carrying the C/G or C/C genotypes, significantly increased UC risk was found for individuals carrying the G/G genotype. Those carrying the G/G genotype had a significantly increased UC risk compared with those with C/G or C/C genotypes. Patients with UC carrying the G/G genotype had a greater prevalence of muscle-invading (stage T2-T4), high-grade (G2) tumor and immunohistochemicaly overexpressed survivin compared with those carrying the C/G or C/C genotypes. CONCLUSIONS: G/G genotype of the -31C/G polymorphism might be a risk factor for UC development.

Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens.

Allergy to rodent proteins is common among laboratory animal workers. Sensitive methods to measure exposure to these allergens have been developed. These assays are, however, expensive, time-consuming, and require a laboratory facility and methodological expertise. A simple method to screen for allergen spread, or to test whether hygiene standards are maintained, would be useful. Lateral flow immunoassays (LFIAs) are especially suited for field settings; the tests are simple and results are visible within minutes. LFIAs were developed for detection of the rodent urinary allergens Mus m 1 and Rat n 1. Pilot studies were performed in animal facilities in three countries using both extracts from airborne dust samples and samples collected by wiping surfaces. For comparison and determination of sensitivity, the concentrations of rodent urinary allergens in the samples were also measured using enzyme immunoassays (EIAs). The LFIAs for rat and mouse urinary allergens had a detection limit of 31 pg allergen per mL in a buffer system with purified allergen standards. Results of environmental dust extracts tested in LFIAs correlated well with levels obtained using EIAs. Spread of rodent allergens, or non-adherence to hygiene around laboratory animal facilities, may aggravate rodent allergy. Using a simple, sensitive one-step assay, allergens can be detected to prevent allergen exposure. The results reveal that the rapid assays are suited for on-site demonstration of exposure to rodent allergens, and thus, useful in occupational hygiene practice.

A facile nanoparticle immunoassay for cancer biomarker discovery.

BACKGROUND: Gold nanoparticles (AuNPs) scatter light intensely at or near their surface plasmon wavelength region. Using AuNPs coupled with dynamic light scattering (DLS) detection, we developed a facile nanoparticle immunoassay for serum protein biomarker detection and analysis. A serum sample was first mixed with a citrate-protected AuNP solution. Proteins from the serum were adsorbed to the AuNPs to form a protein corona on the nanoparticle surface. An antibody solution was then added to the assay solution to analyze the target proteins of interest that are present in the protein corona. The protein corona formation and the subsequent binding of antibody to the target proteins in the protein corona were detected by DLS. RESULTS: Using this simple assay, we discovered multiple molecular aberrations associated with prostate cancer from both mice and human blood serum samples. From the mice serum study, we observed difference in the size of the protein corona and mouse IgG level between different mice groups (i.e., mice with aggressive or less aggressive prostate cancer, and normal healthy controls). Furthermore, it was found from both the mice model and the human serum sample study that the level of vascular endothelial growth factor (VEGF, a protein that is associated with tumor angiogenesis) adsorbed to the AuNPs is decreased in cancer samples compared to non-cancerous or less malignant cancer samples. CONCLUSION: The molecular aberrations observed from this study may become new biomarkers for prostate cancer detection. The nanoparticle immunoassay reported here can be used as a convenient and general tool to screen and analyze serum proteins and to discover new biomarkers associated with cancer and other human diseases.

A functional nuclear epidermal growth factor receptor, SRC and Stat3 heteromeric complex in pancreatic cancer cells.

Evidence is presented for the nuclear presence of a functional heteromeric complex of epidermal growth factor (EGFR), Src and the Signal Transducer and Activator of Transcription (Stat)3 proteins in pancreatic cancer cells. Stat3 remains nuclear and associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src, demonstrating the resistance of the complex to the modulation of EGFR or Src alone. Significantly, chromatin immunoprecipitation (ChIP) analyses reveal the nuclear EGFR, Src and Stat3 complex is bound to the c-Myc promoter. The siRNA knockdown of EGFR or Src, or the pharmacological inhibition of Stat3 activity only marginally suppressed c-Myc expression. By contrast, the concurrent modulation of Stat3 and EGFR, or Stat3 and Src, or EGFR and Src strongly suppressed c-Myc expression, demonstrating that the novel nuclear heteromeric complex intricately regulates the c-Myc gene. The prevalence of the transcriptionally functional EGFR, Src, and Stat3 nuclear complex provides an additional and novel mechanism for supporting the pancreatic cancer phenotype and explains in part the insensitivity of pancreatic cancer cells to the inhibition of EGFR, Src or Stat3 alone.

The effect of pH on the activity of soluble peroxidase in needles of Serbian spruce (Picea omorika (Panc.) Purkinye): application of a mathematical model.

We studied the dependence of peroxidase (POD) activity on pH in crude extract of Picea omorika (Panc.) Purkinye needles and in its acidic and basic fractions, obtained by ion exchange chromatography. Nonlinear regression was applied on the activity data with pH as the explaining variable, using the Levenberg-Marquardt algorithm. Studying crude extract at three different temperatures, the shape of the simulated activity/pH dependences indicated an existence of two components, which was confirmed by mathematical modeling. The kinetic parameters Act0, KEH and KEOH of both components are presented. The curves and pH optima shifted under increasing temperatures towards lower pH values, which was verified after decomposition. Nonlinear regression detected the presence of two components for both fractions, and there is no considerable difference between their pH optima. Our results show for the first time that the sum of components, each described by the mathematical model employed, can be used to explain the complex pH-related POD activity in the extract with two or more enzyme forms simultaneously active.

A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study.

The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 microg/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study.

Kinetic parameters for thermal inactivation of soluble peroxidase from needles of Serbian spruce Picea omorika (Pancic) Purkyne.

Thermal inactivation of peroxidase (POD) in an extract of Picea omorika (Pancic) Purkyne needles initiated by heat treatment was studied. This is the first study of this kind on a conifer species. Non-linear regression analysis was applied on the inactivation rate data, combining Mitscherlich and Arrhenius equations, treating time and temperature simultaneously as explaining variables. We determined the inactivation rate constant k, the Arrhenius energy of inactivation E and the remaining activity C(min) for the crude extract and for separated acidic and basic enzyme fractions, as well as for individual isoenzymes separated electrophoretically. A comparison of inactivation parameters for acidic and basic fractions shows that the thermal inactivation rate of the basic fraction is higher. The obtained value of inactivation energy for crude extract was between the values for acidic and basic isoenzyme fractions. One of the three analysed individual isoenzymes was characterised by a lower inactivation rate constant and higher inactivation energy. Another isoenzyme showed considerably higher level of remaining activity compared to the others, which identified it as the most resistant to high temperatures. The acquired values of Arrhenius energy of inactivation for POD in crude extract were intermediate, considering a range of POD values for various other plant species.

Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay.

BACKGROUND: Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with direct on-site demonstration of a bioallergen exposure hazard. OBJECTIVE: In a field study, we evaluated a recently developed LFIA for fungal alpha-amylase, an important bakery allergen. METHODS: Airborne and surface dust (wipe) samples and samples from flours and baking additives used at the workplace were collected in 5 industrial bakeries and tested in the LFIA for fungal amylase. For comparison, amylase was measured in sample eluates with the reference EIA method. RESULTS: Sensitivity of the LFIA was 1 to 10 ng/mL, and of EIA, approximately 25 pg/mL. In LFIA, most flour samples, 84% of wipe samples, 26% of personal airborne dust, and none of the 26 ambient air dust samples produced a visible reaction. Wipe samples from dough-making areas and flour samples gave the strongest reactions. All extracts with >5 ng allergen per milliliter showed a positive LFIA reaction. CONCLUSION: The LFIA for fungal amylase is an easy and rapid method to demonstrate the allergen directly at the worksite in less than 10 to 20 minutes. Similar LFIA methods may be used for other occupational allergens in other work environments. CLINICAL IMPLICATIONS: Lateral flow immunoassays for occupational allergens may be of great value in occupational hygiene surveys to demonstrate directly to workers and supervisors the hazards of work-related bioallergen exposure.

A rapid lateral flow immunoassay for the detection of fungal alpha-amylase at the workplace.

Fungal alpha-amylase is a flour supplement which is added to improve the quality of bakery products. Various studies have shown that exposure to this enzyme is an important risk factor for the development of bakers' allergy and this allergy is reported to be one of the most frequent causes of occupational asthma. A rapid assay was developed to monitor exposure to occupational allergens directly at the workplace. The sensitivity of the developed assay is 0.32 ng amylase mL(-1) in a buffer system with the commercially available alpha-amylase preparation Fungamyl 1600S as the standard. Initial validation tests (n = 33) were performed with airborne and settled dust from an industrial bakery. The new lateral flow immunoassay detected amylase in 22 of the 26 samples regarded as positive in an enzyme immunoassay, and was negative for all seven enzyme immunoassay-negative samples, while the four lateral flow immunoassay-negative/enzyme immunoassay-positive samples all had levels below 2 ng mL(-1). The sensitivity of 2 ng mL(-1) of the amylase lateral flow immunoassay is sufficient for first screening purposes and, therefore, this simple and rapid assay may allow direct on-site demonstration of work-related hazards of bio-allergen exposure. This would be particularly useful in occupational hygiene practice, especially in traditional or small-scale bakeries which lack the technological skills for testing the exposure to respiratory allergens.

Nasal air sampling for measuring inhaled wheat allergen in bakeries with and without facemask use.

OBJECTIVE: Occupational asthma caused by flour is common in bakers. We applied novel intranasal air samplers (INAS) to assess wheat allergen exposure and evaluate respiratory protection in bakeries. METHODS: Two models of INAS (INAS-M1 and INAS-M2) were compared with simultaneous personal air sampling of inhalable dust, both with and without facemasks. Wheat allergen levels were measured using a sensitive sandwich enzyme immunoassay. Allergenic particles were immunostained for microscopic visualization. RESULTS: Personal air sampling correlated well with INAS-M1 (r = 0.89) and INAS-M2 (r = 0.75). INAS-M2 collected particles more effectively than INAS-M1. Facemasks reduced inhalation of wheat allergen by 96% and 93% measured using INAS-M1 and INAS-M2, respectively. CONCLUSIONS: Nasal air sampling can complement personal air sampling to measure short-term exposure and evaluate respiratory protection. To prevent baker's asthma, facemasks may be an effective solution in addition to improving workplaces.

Airborne exposure to wheat allergens: optimised elution for airborne dust samples.

Well-validated methods for measuring airborne occupational allergens are essential for effective control and reduction of allergen exposures. For wheat flour allergens, specific immunoassays are available, but there is a need for optimisation and standardization of sample processing procedures. Wheat flour allergen elution and storage were studied using airborne dust samples collected in bakeries with a new parallel sampler. Forty-eight series of 9 parallel filters were subjected to extraction procedures varying in elution medium, shaking method, extraction vial, and centrifugation speed. Wheat allergens were measured with enzyme immunoassays, and the effect of various procedures evaluated by mixed regression analyses. The stability of the eluted allergens was assessed after storage for 20 days and 4 months at -20 degrees C, in the presence or absence of casein in the medium. Only the type of elution medium had significant effects on allergen recovery: addition of Tween-20 resulted in 3- to 100-fold increased levels, an effect that was most pronounced at low concentrations. Allergen levels in extracts were stable for at least 4 months at -20 degrees C, irrespective of the presence of casein in the medium. Addition of Tween-20 to the elution medium is essential for optimal extraction of wheat allergen. The recommended procedure further includes the use of conventional polystyrene tubes, simple shaking methods, and centrifugation after extraction. Wheat dust extracts in PBS-Tween can be stored frozen for at least 4 months, and addition of a stabilising protein is not required.

Characterization of enzymatically synthesized diferulate.

Horseradish peroxidase was used to synthesize diferulates by a procedure in which ethyl ferulate was used as substrate. Four different forms were obtained, of which two dominant were the 5-5' and 8-5' diferulate. Fluorescence emission spectra of the diferulates (excited at 284 nm) indicate that they contain two chromophores, as opposed to the substrate molecule. Fluorescence excitation spectra with emission at 417 nm further demonstrate the difference between the synthesized diferulates and starting substrates.