Higher-Level Steatosis Is Associated with a Greater Decrease in Metabolic Dysfunction-Associated Steatoic Liver Disease after Eight Weeks of a Very Low-Calorie Ketogenic Diet (VLCKD) in Subjects Affected by Overweight and Obesity.

The most common form of chronic liver disease, recently defined as MASLD, is strongly linked to obesity and metabolic syndrome. Lifestyle changes are part of MASLD prevention. The very low-calorie ketogenic diet (VLCKD) is a useful option for treating MASLD and reducing liver steatosis in patients with obesity. We assessed whether a greater degree of steatosis could have a positive or negative impact on how well 8 weeks of using the VLCKD improve steatosis and fibrosis in a patient population of overweight and obese individuals. Anthropometric parameters, along with changes in hormone and metabolic biomarkers, were also assessed both before and after the dietary change. The study population included 111 overweight (14.41%) or obese subjects (85.59%) aged between 18 and 64 years; the 75 women and 36 men involved were not taking any medicine. In both the raw (0.37 95% CI 0.21; 0.52) and the multivariate models (model a: 0.439 95% CI 0.26; 0.62; model b: 0.437 95% CI 0.25; 0.63), there was a positive and statistically significant correlation between the CAP delta value and the CAP before using the VLCKD. Additionally, the liver stiffness delta was found to be positively and statistically significantly correlated with liver stiffness before the use of the VLCKD in both models: the multivariate model (model a: 0.560 95% CI 0.40; 0.71; model b: 0.498 95% CI 0.34; 0.65) and the raw model (0.52 95% CI 0.39; 0.65). Systolic and diastolic blood pressure, insulin resistance (measured by HOMA-IR), insulin, HbA1c, fasting blood glucose, total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides, BMI, waist circumference, and fat mass, were all decreased (p < 0.001) following the use of the VLCKD. However, following the use of the VLCKD, there was an increase in vitamin D levels. (p < 0.001). We found that using the VLCKD for 8 weeks has a greater effect on improving steatosis and fibrosis in subjects who initially have more severe forms of these conditions.

Metabolomic Profiling of Obese Patients with Altered Intestinal Permeability Undergoing a Very Low-Calorie Ketogenic Diet.

A healthy intestinal permeability facilitates the selective transport of nutrients, metabolites, water, and bacterial products, involving cellular, neural, hormonal, and immune factors. An altered intestinal permeability indicates pathologic phenotypes and is associated with the exacerbation of obesity and related comorbidities. To investigate the impact of altered permeability in obese patients undergoing a calorie-restrictive dietary regimen (VLCKD), we collected urinary and fecal samples from obese patients with both normal and altered permeability (determined based on the lactulose/mannitol ratio) before and after treatment. The analysis of volatile organic compounds (VOCs) aids in understanding the metabolites produced by the intestinal microbiota in this unique ecological niche. Furthermore, we examined clinical and anthropometric variables from the cohort and compared them to significant VOC panels. Consequently, we identified specific markers in the metabolomics data that differentiated between normal and altered profiles before and after the diet. These markers indicated how the variable contribution specifically accounted for interleukins and lipopolysaccharides (LPS). The targeted metabolomics experiment detected no differences in measured short-chain fatty acids (SCFA). In summary, our study evaluated metabolomic markers capable of distinguishing low-grade inflammation conditions, exacerbated in more advanced stages of obesity with altered intestinal permeability.

Effects of an Eight Week Very Low-Calorie Ketogenic Diet (VLCKD) on White Blood Cell and Platelet Counts in Relation to Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) in Subjects with Overweight and Obesity.

Obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) are frequently associated conditions characterized by low-grade inflammation. Very low-calorie ketogenic diet (VLCKD) strategies are commonly used to simultaneously obtain weight loss and an improvement of liver steatosis. We evaluated the efficacy of 8 weeks' VLCKD in decreasing the white blood cell (WBC) and platelet (PLT) counts, as well as liver steatosis and fibrosis, diagnosed using transient elastography (FibroScan). Metabolic and anthropometric parameters commonly associated with MASLD were also evaluated. This study included 87 participants; 58 women and 29 men aged between 18 and 64 years with overweight (18%) or obesity (82%), but not taking any medication. Anthropometric measurements, bioimpedance analysis, and biochemical assays were performed before and after the dietary intervention. BMI (kg/m2) (p-value < 0.001), waist circumference (cm) (p-value < 0.001), and fat mass (kg) (p-value < 0.001) were significantly decreased following VLCKD. After VLCKD, the FibroScan parameter CAP (db/m), which measures the accumulation of fatty liver, significantly decreased (p-value < 0.001), as did liver stiffness (kPA), the FibroScan parameter quantifying liver fibrosis (p-value < 0.05). Seemingly, WBC (p-value < 0.001) and PLT (p-value < 0.001) counts were lowered by VLCKD in the whole group; however, the decrease in WBC and platelet counts were significant only in patients with steatosis (CAP ≥ 215 dB/m). Fasting blood glucose (p-value < 0.001), insulin (p-value < 0.001), HbA1c (p-value < 0.001), triglycerides (p-value < 0.001), total cholesterol (p-value < 0.001), LDL-cholesterol (p-value < 0.001), HDL-cholesterol (p-value < 0.001); γGT (p-value < 0.001) blood levels and insulin resistance (as measured by HOMAIR) (p-value < 0.001); and systolic (p-value < 0.001), and diastolic (p-value < 0.001) blood pressure levels, were all significantly lower after VLCKD. In contrast, blood levels of vitamin D were higher following the diet (p-value < 0.001). We conclude that treating subjects with overweight and obesity with VLCKD is followed by a simultaneous reduction in WBCs and platelets, the expression of low-grade inflammation, and of liver steatosis and fibrosis. Therefore, we can hypothesize that VLCKD decreases general and liver low-grade inflammation, thus improving liver health.

Effects of a Very-Low-Calorie Ketogenic Diet on the Fecal and Urinary Volatilome in an Obese Patient Cohort: A Preliminary Investigation.

Several recent studies deepened the strong connection between gut microbiota and obesity. The effectiveness of the very-low-calorie ketogenic diet (VLCKD) has been measured in terms of positive impact on the host homeostasis, but little is known of the modification exerted on the intestinal metabolome. To inspect this complex relationship, we analyzed both fecal and urinary metabolome in terms of volatile organic compounds (VOCs) by the GC-MS method in 25 obese patients that were under VLCKD for eight weeks. Partial least square discriminant analysis evidenced specific urinary and fecal metabolites whose profile can be considered a signature of a partial restore toward the host eubiosis. Specifically, among various keystone VOCs, the decreased concentration of four statistically significant fecal esters (i.e., propanoic acid pentyl ester, butanoic acid hexyl ester, butanoic acid pentyl ester, and pentanoic acid butyl ester) supports the positive effect of VLCKD treatment. Our pilot study results suggest a potential positive effect of VLCKD intervention affecting fecal and urinary volatilome profiles from obese patients. Meta-omics techniques including the study of genes and transcripts will help in developing new interventions useful in preventing or treating obesity and its associated health problems.

Age-Related Dietary Habits and Blood Biochemical Parameters in Patients with and without Steatosis-MICOL Cohort.

BACKGROUND: Steatosis is now the most common liver disease in the world, present in approximately 25% of the global population. The aim of this study was to study the association between food intake and liver disease and evaluate the differences in blood parameters in age classes and steatosic condition. METHODS: The present study included 1483 participants assessed in the fourth recall of the MICOL study. Patients were subdivided by age (65 years) and administered a validated food frequency questionnaire (FFQ) with 28 food groups. RESULTS: The prevalence of steatosis was 55.92% in the adult group and 55.88% in the elderly group. Overall, the results indicated many statistically significant blood parameters and dietary habits. Analysis of food choices with a machine learning algorithm revealed that in the adult group, olive oil, grains, processed meat, and sweets were associated with steatosis, while the elderly group preferred red meat, dairy, seafood, and fruiting vegetables. Furthermore, the latter ate less as compared with the adult group. CONCLUSIONS: Many differences were found between the two age groups, both in blood parameters and food intake. The random forest also revealed different foods predicted steatosis in the two groups. Future analysis will be useful to understand the molecular basis of these differences and how different food intake causes steatosis in people of different ages.

The Effects of a Very-Low-Calorie Ketogenic Diet on the Intestinal Barrier Integrity and Function in Patients with Obesity: A Pilot Study.

The very-low-calorie ketogenic diet (VLCKD) is effective and safe for obese individuals, but limited information exists on its impact on the intestinal barrier. This study analyzed the effects of 8 weeks of VLCKD on 24 obese patients (11M/13F). Carbohydrate intake was fixed at 20-50 g/day, while protein and lipid intake varied from 1-1.4 g/kg of ideal body weight and 15-30 g per day, respectively. Daily calorie intake was below 800 kcal. The lactulose-mannitol absorption test assessed small intestinal permeability. Multiple markers, such as serum and fecal zonulin, fatty acid-binding protein, diamine oxidase concentrations, urinary dysbiosis markers (indican and skatole), and circulating lipopolysaccharide levels, were analyzed. Inflammation markers (serum interleukin 6, 8, 10, and tumor necrosis factor-α concentrations) were also evaluated. The results showed significant reductions in weight, BMI, and waist circumference post-diet. However, the lactulose-mannitol ratio increased by 76.5%, and a significant increase in dysbiosis markers at the end of the diet occurred. This trend was particularly evident in a subgroup of patients. Despite initial benefits, the VLCKD might negatively affect the intestinal barrier function in obese patients, potentially worsening their compromised intestinal balance.

Food Network Analysis in Non-Obese Patients with or without Steatosis.

BACKGROUND: Steatosis is the most common liver disease worldwide and the leading cause of liver-associated morbidity and mortality. The aim of this study was to explore the differences in blood parameters and dietary habits in non-obese patients with and without steatosis. METHODS: The present study included 987 participants with BMI < 30, assessed in the fourth recall of the MICOL study. Patients were divided by steatosis grade, and a validated food frequency questionnaire (FFQ) with 28 food groups was administered. RESULTS: The prevalence of non-obese participants with steatosis was 42.86%. Overall, the results indicated many statistically significant blood parameters and dietary habits. Analysis of dietary habits revealed that non-obese people with or without steatosis had similar dietary habits, although higher daily amounts of red meat, processed meat, ready meals, and alcohol were recorded in participants with liver disease (p < 0.05). CONCLUSIONS: Many differences were found in non-obese people with and without steatosis, but in light of a network analysis, the two groups demonstrated similar dietary habits, proving that pathophysiological, genetic, and hormonal patterns are probably the basis of their liver status, regardless of weight. Future genetic analyses will be performed to analyze the expression of genes involved in the development of steatosis in our cohort.

In Vitro Evaluation of the Antibacterial Activity of the Peptide Fractions Extracted from the Hemolymph of Hermetia illucens (Diptera: Stratiomyidae).

Antimicrobial peptides (AMPs) are a chemically and structurally heterogeneous family of molecules produced by a large variety of living organisms, whose expression is predominant in the sites most exposed to microbial invasion. One of the richest natural sources of AMPs is insects which, over the course of their very long evolutionary history, have adapted to numerous and different habitats by developing a powerful innate immune system that has allowed them to survive but also to assert themselves in the new environment. Recently, due to the increase in antibiotic-resistant bacterial strains, interest in AMPs has risen. In this work, we detected AMPs in the hemolymph of Hermetia illucens (Diptera, Stratiomyidae) larvae, following infection with Escherichia coli (Gram negative) or Micrococcus flavus (Gram positive) and from uninfected larvae. Peptide component, isolated via organic solvent precipitation, was analyzed by microbiological techniques. Subsequent mass spectrometry analysis allowed us to specifically identify peptides expressed in basal condition and peptides differentially expressed after bacterial challenge. We identified 33 AMPs in all the analyzed samples, of which 13 are specifically stimulated by Gram negative and/or Gram positive bacterial challenge. AMPs mostly expressed after bacterial challenge could be responsible for a more specific activity.

The Replacement of Only One Portion of Starchy Carbohydrates with Green Leafy Vegetables Regresses Mid and Advanced Stages of NAFLD: Results from a Prospective Pilot Study.

The gold standard treatment for NAFLD is weight loss and lifestyle interventions, which require a diet enriched in fiber and reduced in sugars and saturated fats. Fibres may be advantageous for NAFLD patients since they reduce and slow the absorption of carbohydrates, lipids, and proteins, lowering the energy density of the meal and increasing their sense of satiety. Furthermore, the polyphenol content and other bioactive compounds of vegetables have antioxidant and anti-inflammatory properties preventing disease progression. The aim of this study is to ascertain the effects of a diet enriched by green leafy vegetables and with a moderate restriction of carbohydrate intake in patients with NAFLD over a three month period. Among the forty patients screened, twenty four patients completed the clinical trial consisting of swapping one portion of carbohydrate-rich food for one portion of green leafy vegetables, and liver and metabolic markers of NAFLD were evaluated. All patients underwent routine blood tests, anthropometric measurements, bioelectrical impedance analysis, fibroscan, and fatty liver index (FLI) evaluation before and at the end of the study. The population under study (n = 24) had a median age of 47.5 (41.5-52.5) years and included mainly women (70.8%). We found that FLI, which is used to predict fatty liver (73 (33-89) vs. 85 (54-95), p < 0.0001) and the FAST score, which is a fibroscan-derived parameter identifying patients at risk of progressive NASH (0.03 (0.02-0.09) vs. 0.05 (0.02-0.15), p = 0.007), were both improved after changes in diet. The BMI (33.3 (28.6-37.3) vs. 35.3 (31.2-39.0), p < 0.0001), WC (106.5 (95.0-112.5) vs. 110.0 (103.0-124.0), p < 0.0001), neck circumference (38.0 (35.0-41.5) vs. 39.5 (38.0-42.5), p < 0.0001), fat mass (32.3 (23.4-40.7) vs. 37.9 (27.7-43.5), p < 0.0001), and extracellular water (17.3 (15.2-20.8) vs. 18.3 (15.9-22.7), p = 0.03) were also all significantly lower after three months of diet. Metabolic parameters linked to NAFLD decreased: HbA1c (36.0 (33.5-39.0) vs. 38.0 (34.0-40.5), p = 0.01), triglycerides (72 (62-90) vs. 90 (64-132), p = 0.03), and the liver markers AST (17 (14-19) vs. 18 (15-27), p = 0.01) and γGT (16 (13-20) vs. 16 (14-27), p = 0.02). In conclusion, replacing only one portion of starchy carbohydrates with one portion of vegetables for a three month period is sufficient to regress, at least in part, both mid and advanced stages of NAFLD. This moderate adjustment of lifestyle habits is easily achievable.

Gender and Liver Steatosis Discriminate Different Physiological Patterns in Obese Patients Undergoing Bariatric Surgery: Obesity Center Cohort.

BACKGROUND: Obesity is a major public health problem worldwide. Bariatric surgery can reduce body weight, and it is one of the better ways to improve metabolic disease and lifestyle. The aim of this study was to explore a new cohort of patients with obesity and evaluate the gender differences and the steatosis status within the gender group. METHODS: A cohort of 250 adult obese patients with BMI ≥ 30 and age >18 years, eligible for gastric bariatric surgery at Pineta Grande Hospital, Castel Volturno (Italy) was studied. RESULTS: The prevalence in women was higher (72.40%) than men (27.60%). Overall, results indicated many statistically significant gender differences in hematological and clinical parameters. Analysis of the subcohorts based on the severity of steatosis revealed differences of this condition between the genders. Steatosis was more prevalent in the male subcohort, but female patients revealed greater within-group differences. CONCLUSIONS: Many differences were found not only in the total cohort but also between the gender subcohorts, both in the presence and absence of steatosis. We can conclude that the pathophysiological, genetic, and hormonal patterns affecting these patients delineate different individual profiles.

Corrigendum: Associations between serum biomarkers and non-alcoholic liver disease: results of a clinical study of Mediterranean patients with obesity.

[This corrects the article DOI: 10.3389/fnut.2022.1002669.].

PEGylated Liposomes Loaded with Carbamate Inhibitor ANP0903 Trigger Apoptosis by Enhancing ER Stress in HepG2 Cancer Cells.

Liver cancer is one of the most common causes of cancer death worldwide. In recent years, substantial progress has been made in the development of systemic therapies, but there is still the need for new drugs and technologies that can increase the survival and quality of life of patients. The present investigation reports the development of a liposomal formulation of a carbamate molecule, reported as ANP0903, previously tested as an inhibitor of HIV-1 protease and now evaluated for its ability to induce cytotoxicity in hepatocellular carcinoma cell lines. PEGylated liposomes were prepared and characterized. Small, oligolamellar vesicles were produced, as demonstrated by light scattering results and TEM images. The physical stability of the vesicles in biological fluids was demonstrated in vitro, alongside the stability during storage. An enhanced cellular uptake was verified in HepG2 cells treated with liposomal ANP0903, resulting in a greater cytotoxicity. Several biological assays were performed to elucidate the molecular mechanisms explaining the proapoptotic effect of ANP0903. Our results allow us to hypothesize that the cytotoxic action in tumor cells is probably due to the inhibition of the proteasome, resulting in an increase in the amount of ubiquitinated proteins within the cells, which in turn triggers activation of autophagy and apoptosis processes, resulting in cell death. The proposed liposomal formulation represents a promising approach to deliver a novel antitumor agent to cancer cells and enhance its activity.

The Effects of Eight Weeks' Very Low-Calorie Ketogenic Diet (VLCKD) on Liver Health in Subjects Affected by Overweight and Obesity.

Very low-calorie ketogenic diets (VLCKD) are widely employed in successful weight-loss strategies. Herein, we evaluated the efficacy and safety of a VLCKD on non-alcoholic fatty liver disease (NAFLD) and parameters commonly associated with this condition in overweight and obese subjects who did not take any drugs. This prospective, real-life study included thirty-three participants who followed a VLCKD for 8 weeks. NAFLD was diagnosed using transient elastography (FibroScan). Data on anthropometric measurements, bioimpedance analysis, and biochemical assays were gathered both before and after the dietary intervention. BMI (kg/m2) (from 33.84 ± 6.55 to 30.89 ± 6.38, p < 0.01), waist circumference (cm) (from 106.67 ± 15.51 to 98.64 ± 16.21, p < 0.01), and fat mass (Kg) (from 38.47 ± 12.59 to 30.98 ± 12.39, p < 0.01) were significantly lower after VLCKD. CAP (db/m), the FibroScan parameter quantifying fatty liver accumulation, showed a significant reduction after VLCKD (from 266.61 ± 67.96 to 223 ± 64.19, p < 0.01). After VLCKD, the fatty liver index (FLI), a benchmark of steatosis, also revealed a significant decline (from 62.82 ± 27.46 to 44.09 ± 31.24, p < 0.01). Moreover, fasting blood glucose, insulin, triglycerides, total cholesterol, LDL-cholesterol, ALT, γGT, and FT3 blood concentrations, as well as insulin resistance (quantified by HOMAIR) and systolic and diastolic blood pressure levels, were significantly lower after VLCKD (p < 0.01 for all the parameters). By contrast, HDL-cholesterol, 25 (OH) vitamin D, and FT4 blood concentrations were higher after VLCKD (p < 0.01 for all parameters). The variation (δ) of CAP after VLCKD did not show a correlation with the δ of any other parameter investigated in this study. We conclude that VLCKD is a helpful approach for NAFLD independent of changes in factors commonly associated with NAFLD (obesity, fat mass, insulin resistance, lipids, and blood pressure) as well as vitamin D and thyroid hormone levels.

Prevalence and risk factors for hospital-acquired anemia in internal medicine patients: learning from the "less is more" perspective.

Hospital-acquired anemia is defined as a new-onset anemia in hospitalized patients who have a normal hemoglobin level at admission. Its prevalence is unknown and most studies published on this topic have been conducted in intensive care unit patients with limited applicability to less acute settings, such as internal medicine wards. We conducted a retrospective study and enrolled 129 patients who were admitted to an Internal Medicine Unit between October 2021 and February 2022. The median value of phlebotomy during hospitalization was 46 ml (IQR 30-72 ml), whereas the median length of hospital stay was 9 days (IQR 5-13 days). The median value of hemoglobin reduction was -0.63 g/dl (p < 0.001) and the maximum value of drop in hemoglobin value was -2.6 g/dl. All patients who experienced a phlebotomy > 85 ml had a hemoglobin reduction > 0.6 g/dl. 20.9% of patients developed anemia during the hospital stay (7% moderate and 13.9% mild). No cases of severe anemia were observed. The volume of blood drawn during the hospital stay and the Hb value on admission were the only two variables statistically associated with the development of anemia, whereas gender, age, and chronic diseases, such as diabetes, history of cancer, or heart failure, were not. Strategies, such as elimination of unnecessary laboratory tests and the use of smaller tubes for blood collection, are needed to reduce the volume of iatrogenic blood loss and avoid blood wastage occurring during hospitalization in internal medicine patients.

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y.

Up-regulated Gene clone 7 (URG7) is a protein localized in the endoplasmic reticulum (ER) and overexpressed in liver cells upon hepatitis B virus (HBV) infection. Its activity has been related to the attenuation of ER stress resulting from HBV infection, promoting protein folding and ubiquitination and reducing cell apoptosis overall. While the antiapoptotic activity of URG7 in HBV-infected cells may have negative implications, this effect could be exploited positively in the field of proteinopathies, such as neurodegenerative diseases. In this work, we aimed to verify the possible contribution of URG7 as a reliever of cellular proteostasis alterations in a neuronal in vitro system. Following tunicamycin-induced ER stress, URG7 was shown to modulate different markers of the unfolded protein response (UPR) in favor of cell survival, mitigating ER stress and activating autophagy. Furthermore, URG7 promoted ubiquitination, and determined a reduction in protein aggregation, calcium release from the ER and intracellular ROS content, confirming its pro-survival activity. Therefore, in light of the results reported in this work, we hypothesize that URG7 offers activity as an ER stress reliever in a neuronal in vitro model, and we paved the way for a new approach in the treatment of neurodegenerative diseases.

Once-Weekly Subcutaneous Semaglutide Improves Fatty Liver Disease in Patients with Type 2 Diabetes: A 52-Week Prospective Real-Life Study.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is commonly observed in patients with type 2 diabetes (T2D). Semaglutide, a glucagon-like peptide 1 receptor agonist, may have a therapeutic role by targeting common mechanisms involved in the pathophysiology of T2D and NAFLD. The study aimed to assess the effectiveness of Semaglutide on NAFLD in patients with T2D. METHODS: Forty-eight patients were treated with subcutaneous Semaglutide in add-on to metformin for 52 weeks. After the baseline visit (T0), follow-up was scheduled quarterly (T3, and T6) and then at 12 months of therapy (T12). During each visit, body composition was analyzed by phase-sensitive bio-impedance, and NAFLD was diagnosed and staged by Ultrasound (US) imaging. Surrogate biomarkers of NAFLD were also calculated and followed over time. RESULTS: A significant decrease in anthropometric and glucometabolic parameters, insulin resistance, liver enzymes, and laboratory indices of hepatic steatosis was observed during treatment. Similarly, fat mass and visceral adipose tissue (VAT) decreased over time more than skeletal muscle and free-fat mass. US-assessed VAT thickness and the 12-point steatosis score also declined at T3 up to T12. Liver steatosis improved in most patients (70%), showing a reduction by at least one class in the semiquantitative US staging. CONCLUSION: Besides glucose control and body composition improvements, Semaglutide was effective in ameliorating the clinical appearance and severity of NAFLD in T2D patients.

Associations between serum biomarkers and non-alcoholic liver disease: Results of a clinical study of Mediterranean patients with obesity.

Background: Transient elastography is an ultrasound-based method to detect non-alcoholic fatty liver disease (NAFLD). Despite the simultaneously rising prevalence of fatty liver and metabolic disease, further information about metabolic risk indicators of fatty liver is still necessary. Methods: A Southern Italian population sample with obesity (N = 87) was cross-sectionally explored for associations among the presence of NAFLD, assessed by FibroScan, and clinical, biochemical and anthropometric parameters. Inclusion criteria were age >18 years, BMI ≥ 25 kg/m2, no ongoing supplemental or drug therapy, including oral contraceptives or osteoporosis medications; exclusion criteria were pregnancy, endocrinological diseases, cardiovascular diseases, neoplasia, renal or hepatic failure, hereditary thrombocytopenia, hepatitis B (HBV) or hepatitis C virus (HCV) infection, and excess alcohol consumption. Results: The study sample featured a female predominance (67%, N = 60), age range 18-64 years, and 40% prevalence of NAFLD, in accordance with the fibroscan-measured controlled attenuation parameter (CAP) threshold value above 302 dB/m. Males were slightly more frequently affected by NAFLD (51.4% vs. 48.6%, p = 0.01). Insulin levels, insulin resistance (quantified by HOMA-IR), diastolic blood pressure, BMI, visceral adipose tissue (VAT), and waist circumference were significantly higher in the NAFLD subset compared to their counterparts (p < 0.01, p < 0.01, p = 0.05, p < 0.01, p < 0.01, p < 0.01, respectively). Uric acid (p < 0.01) also showed a positive trend in the NAFLD group. Other liver steatosis parameters, measured by stiffness (p < 0.01), fatty liver index (FLI) (p < 0.01) and FibroScan-AST (FAST) (p < 0.01), were also significantly greater in the NAFLD group. In three nested linear regression models built to assess associations between CAP values and serum uric acid levels, a single unit increase in uricemia indicated a CAP increase by 14 dB/m, after adjusting for confounders (coefficient: 14.07, 95% CI 0.6-27.54). Conclusions: Clinical-metabolic screening for NAFLD cannot ignore uricemia, especially in patients with obesity.

Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid.

OBJECTIVES: The core cerebrospinal fluid (CSF) biomarkers; total tau (tTau), phospho-tau (pTau), amyloid ß 1-42 (Aß 1-42), and the Aß 1-42/Aß 1-40 ratio have transformed Alzheimer's disease (AD) research and are today increasingly used in clinical routine laboratories as diagnostic tools. Fully automated immunoassay instruments with ready-to-use assay kits and calibrators has simplified their analysis and improved reproducibility of measurements. We evaluated the analytical performance of the fully automated immunoassay instrument LUMIPULSE G (Fujirebio) for measurement of the four core AD CSF biomarkers and determined cutpoints for AD diagnosis. METHODS: Comparison of the LUMIPULSE G assays was performed with the established INNOTEST ELISAs (Fujirebio) for hTau Ag, pTau 181, ß-amyloid 1-42, and with V-PLEX Plus Aß Peptide Panel 1 (6E10) (Meso Scale Discovery) for Aß 1-42/Aß 1-40, as well as with a LC-MS reference method for Aß 1-42. Intra- and inter-laboratory reproducibility was evaluated for all assays. Clinical cutpoints for Aß 1-42, tTau, and pTau was determined by analysis of three cohorts of clinically diagnosed patients, comprising 651 CSF samples. For the Aß 1-42/Aß 1-40 ratio, the cutpoint was determined by mixture model analysis of 2,782 CSF samples. RESULTS: The LUMIPULSE G assays showed strong correlation to all other immunoassays (r>0.93 for all assays). The repeatability (intra-laboratory) CVs ranged between 2.0 and 5.6%, with the highest variation observed for ß-amyloid 1-40. The reproducibility (inter-laboratory) CVs ranged between 2.1 and 6.5%, with the highest variation observed for ß-amyloid 1-42. The clinical cutpoints for AD were determined to be 409 ng/L for total tau, 50.2 ng/L for pTau 181, 526 ng/L for ß-amyloid 1-42, and 0.072 for the Aß 1-42/Aß 1-40 ratio. CONCLUSIONS: Our results suggest that the LUMIPULSE G assays for the CSF AD biomarkers are fit for purpose in clinical laboratory practice. Further, they corroborate earlier presented reference limits for the biomarkers.

Two Novel Precursors of the HIV-1 Protease Inhibitor Darunavir Target the UPR/Proteasome System in Human Hepatocellular Carcinoma Cell Line HepG2.

Background: Several pre-clinical and clinical reports suggest that HIV-1 protease inhibitors, in addition to the antiretroviral properties, possess pleiotropic pharmacological effects including anticancer action. Therefore, we investigated the pro-apoptotic activity in tumor cells of two molecules, RDD-19 and RDD-142, which are hydroxyethylamine derivatives' precursors of darunavir and several HIV-1 protease inhibitors. Methods: Three hepatoma cell lines and one non-pathological cell line were treated with RDD-19 and RDD-142, and cell viability was assessed. The expression levels of several markers for ER stress, autophagy, cellular ubiquitination, and Akt activation were quantified in HepG2 cells treated with RDD-19 and RDD-142 to evaluate apoptotic and non-apoptotic cell death. Results: RDD-19 and RDD-142 showed a greater dose-dependent cytotoxicity towards the hepatic tumor cell line HepG2 compared to the non-pathological hepatic cell line IHH. Both molecules caused two types of cell death, a caspase-dependent apoptosis, which was ascertained by a series of biochemical and morphological assays, and a caspase-independent death that was characterized by the induction of ER stress and autophagy. The strong increase of ubiquitinated proteins inside the cells suggested that the target of these molecules could be the proteasome and in silico molecular docking analysis that was used to support the plausibility of this hypothesis. Furthermore, cells treated with the two compounds displayed decreased levels of p-AKT, which interferes with cell survival and proliferation. Conclusions: These findings demonstrate that two compounds, RDD-19 and RDD-142, have pleiotropic effects and that they may represent promising anticancer candidates.