Genetics of fat deposition.

Adipose tissue distribution usually varies among men and women. In men, adipose tissue is known to accumulate in the abdominal region surrounding the visceral organs (android fat distribution) whereas, in women, the accumulation of adipose tissue generally occurs in the gluteal-femoral regions (gynoid fat distribution). In some cases, however, android distribution can be found in women and gynoid distribution can be found in men. The regulation of adipose tissue accumulation involves interaction of a variety of genetic and environmental factors. This review examines genetic factors that cause differential distribution of adipose tissue in different depots of the body, between men and women and between different ethnicities. Genome-wide association studies can be used to identify genetic associations with the distribution and accumulation of adipose tissue. Insight into adipose tissue accumulation and distribution mechanisms could lead to development of personalized interventions for people who develop increased fat mass.

Steroid-converting enzymes in human adipose tissues and fat deposition with a focus on AKR1C enzymes.

Adipocytes express various enzymes, such as aldo-keto reductases (AKR1C), 11ß-hydroxysteroid dehydrogenase (11ß-HSD), aromatase, 5α-reductases, 3ß-HSD, and 17ß-HSDs involved in steroid hormone metabolism in adipose tissues. Increased activity of AKR1C enzymes and their expression in mature adipocytes might indicate the association of these enzymes with subcutaneous adipose tissue deposition. The inactivation of androgens by AKR1C enzymes increases adipogenesis and fat mass, particularly subcutaneous fat. AKR1C also causes reduction of estrone, a weak estrogen, to produce 17ß-estradiol, a potent estrogen and, in addition, it plays a role in progesterone metabolism. Functional impairments of adipose tissue and imbalance of steroid biosynthesis could lead to metabolic disturbances. In this review, we will focus on the enzymes involved in steroid metabolism and fat tissue deposition.

Study of the effects of Lemna minor extracts on human immune cell populations.

OBJECTIVE: Lemna minor is a plant with a huge repertoire of secondary metabolites. The literature indicates that extracts of Lemna minor have antioxidant, antiradical, immunomodulatory and anti-inflammatory properties. The objective of the present study was to find a suitable technique to extract active compounds from this plant and verify whether these extracts have immunomodulatory activity. MATERIALS AND METHODS: We grew L. minor on a standard medium with Gamborg B5 and vitamins. We extracted compounds from the plant by maceration and decoction. The phytochemical profile of the extracts was characterized by chromatography, spectrophotometry, and spectroscopy. The extracts were tested on cultures of mononuclear cells from four human subjects. These cells were pulsed with carboxyfluorescein succinimidyl ester, grown in triplicate in standard culture medium without (control) and with increasing concentrations of Lemna extracts. Flow cytometry was used to evaluate cell death and proliferation of the total mononuclear cell population and of CD4+, CD8+, B cell and monocyte populations. RESULTS: The Lemna extracts were not cytotoxic and did not cause cell necrosis or apoptosis in immune cells. At low concentrations, they induced very limited proliferation of CD4+ cells within 48 hours. At high concentrations, they induced proliferation of CD8+ cells and B lymphocytes within 48 hours. CONCLUSIONS: Unfortunately, we failed to confirm any immunomodulatory activity of Lemna extracts. Growth and death rates of human immune cells were not significantly affected by adding Lemna extracts to the culture medium.

In vitro and clinical studies on the efficacy of α-cyclodextrin and hydroxytyrosol against SARS-CoV-2 infection.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new coronavirus responsible for the current pandemic of coronavirus disease 2019 (COVID-19). This virus attacks cells of the airway epithelium by binding transmembrane angiotensin-converting enzyme 2 (ACE2). Hydroxytyrosol has anti-viral properties. Alpha-cyclodextrin can deplete sphingolipids and phospholipids from cell membranes. The aim of the present experimental study was to evaluate the efficacy of α-cyclodextrin and hydroxytyrosol in improving defenses against SARS-CoV-2 infection in in vitro cell models and humans. PATIENTS AND METHODS: For in vitro experiments on Vero E6 cells, RNA for RT-qPCR analysis was extracted from Caco2 and human fibroblast cell lines. For study in humans, the treatment group consisted of 149 healthy volunteers in Northern Cyprus, considered at higher risk of SARS-CoV-2 infection than the general population. The volunteers used nasal spray containing α-cyclodextrin and hydroxytyrosol for 4 weeks. The control group consisted of 76 healthy volunteers who did not use the spray. RESULTS: RT-qPCR experiments on targeted genes involved in endocytosis showed a reduction in gene expression, whereas cytotoxicity and cytoprotective tests showed that the compounds exerted a protective effect against SARS-CoV-2 infection at non-cytotoxic concentrations. None of the volunteers became positive to SARS-CoV-2 RT-qPCR assay during the 30 days of treatment. CONCLUSIONS: Treatment with α-cyclodextrin and hydroxytyrosol nasal spray improved defenses against SARS-CoV-2 infection and reduced synthesis of viral particles.

Naturally-occurring and cultured bacteriophages in human therapy.

OBJECTIVE: The aim of the study was to show the importance of developing techniques that could exploit the potential of bacteriophages as therapeutics or food supplements. MATERIALS AND METHODS: PubMed database was searched using the following combination of keywords: (bacteriophage) AND (human therapy); (natural bacteriophage) AND (application). RESULTS: The increasing antibiotic resistance of many bacterial strains is making standard antibiotic treatments less effective. Phage therapy provides a non-antibiotic alternative with greater specificity and without harmful effects on the human microbiota. Phages target their specific bacteria, replicate, and then, destroy the host pathogen. Bacteriophages may be administered by several routes, including topical, oral and intravenous. They not only destroy the host pathogen but, in some cases, increase the sensitivity of host bacteria to antibiotics. Various studies have shown that combining phage therapy and antibiotic treatment can be effective against bacterial infections. Clinical trials of phage therapy have shown promising results for various human diseases and conditions. With advances in genetic engineering and molecular techniques, bacteriophages will be able to target a wide range of bacteria. CONCLUSIONS: In the future, phage therapy promises to become an effective therapeutic option for bacterial infections. Since many potentially beneficial bacteriophages can be found in food, supplements containing bacteriophages could be designed to remodel gut microbiota and eliminate pathogenic bacteria. Remodeling of gut microbiota could correct gut dysbiosis. The order of phages known to have these promising activities is Caudovirales, especially the families Siphoviridae and Myoviridae.

Pheromone receptors and their putative ligands: possible role in humans.

Pheromones are ectohormones that play an important role in communication and behavior. Pheromones and pheromone receptor genes are important in mice and other mammals that rely heavily on pheromone cues to survive. Although there is controversy about whether pheromones and pheromone receptor genes have the same importance or are even active in humans, there are some hints that they might have roles in sociosexual behavior and mental disorders. The aim of this qualitative review was to provide an overview of the state of the art regarding pheromones and pheromone receptors in humans and their possible implications in human physiology and pathology. An electronic search was conducted in MEDLINE, PubMed and Scopus databases for articles published in English up to December 2018. The search concerned a possible role of pheromones and pheromone receptors in humans with implications for sociosexual behavior, mental disorders, the menstrual cycle and nutrition. Pheromone communication in humans has not been definitively demonstrated. However, the potential ability of putative pheromones to activate the hypothalamus, which controls the release of many hormones, suggests they could have a role in systemic functions in humans. Future confirmation of the effects of pheromones and pheromone receptors in humans could be useful in the prevention and treatment of various human disorders.

Putative role of Brugada syndrome genes in familial atrial fibrillation.

OBJECTIVE: Familial atrial fibrillation (FAF), a not uncommon arrhythmia of the atrium, is characterized by heritability, early onset and absence of other heart defects. The molecular and genetic basis is still not completely clear and genetic diagnosis cannot be achieved in about 90% of patients. In this study, we present the results of genetic screening by next generation sequencing in affected Russian families. PATIENTS AND METHODS: Sixty subjects (18 probands and 42 relatives) with a clinical diagnosis of FAF were enrolled in the study. Since AF frequently associates with other cardiomyopathies, we included all genes that were known to be associated with these disorders at the time of our study. All probands were therefore systematically screened for 47 genes selected from the literature. RESULTS: Our study revealed that seven variants co-segregated with the clinical phenotype in seven families. Interestingly, four out of six genes and three out of seven variants have already been associated with Brugada syndrome in the literature. CONCLUSIONS: To our knowledge, this is the first report of association of the CACNA1C, CTNNA3, PKP2, ANK2 and SCN10A genes with FAF; it is also the first study in Russian families.

PipeMAGI: an integrated and validated workflow for analysis of NGS data for clinical diagnostics.

OBJECTIVE: We describe how to set up a custom workflow for the analysis of next generation sequencing (NGS) data suitable for the diagnosis of genetic disorders and that meets the strictest standards of quality and accuracy. Our method goes from DNA extraction to data analysis with a computational in-house pipeline. The system was extensively validated using three publicly available Coriell samples, estimating accuracy, sensitivity and specificity. Multiple runs were also made to assess repeatability and reproducibility. MATERIALS AND METHODS: Three different Coriell samples were analyzed in a single run to perform coverage, sensitivity, specificity, accuracy, reproducibility and repeatability analysis. The three samples were analyzed with a custom-made oligonucleotide probe library using Nextera Rapid Capture enrichment technique and subsequently quantified using the Qubit method. Sample quality was verified using a 4200 TapeStation and sequenced on a MiSeq personal sequencer. Analysis of NGS data was then performed with a custom pipeline. RESULTS: The workflow enabled an accurate and precise analysis of NGS data that meets all the requirements of quality and accuracy required by international standards such as ISO15189 and the Association of Molecular Pathology. CONCLUSIONS: The proposed analysis/validation workflow has high assay accuracy, precision and robustness and can, therefore, be used for clinical diagnostic applications.

Taste, olfactory and texture related genes and food choices: implications on health status.

OBJECTIVE: The food choices are due to a mixture of sensory signals including gustatory, olfactory, and texture sensations. The aim of this quality review was to update data about studies concerning genetics of taste, olfactory and texture receptors and their influence on the health status in humans. MATERIALS AND METHODS: An electronic search was conducted in MEDLINE, Pubmed database and Scopus, for articles published in English until December 2018. Two independent researches selected the studies and extracted the data. RESULTS: The review confirms the importance of inter-individual variations in taste, olfactory and texture related genes on food choices and their implications in the susceptibility to nutrition-related conditions such as obesity, dental caries, diabetes, cardiovascular disease, hypertension, hyperlipidemia and cancer. CONCLUSIONS: The knowledge of variants in taste, olfactory and texture related genes can contribute to the prevention of diseases related to unhealthy nutrition. Further studies would be useful to identify other variants in the genes involved in these systems.

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

CREB engages C/EBPδ to initiate leukemogenesis.

cAMP response element binding protein (CREB) is frequently overexpressed in acute myeloid leukemia (AML) and acts as a proto-oncogene; however, it is still debated whether such overactivation alone is able to induce leukemia as its pathogenetic downstream signaling is still unclear. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. Gene expression analysis of CREB-zebrafish revealed a signature of 20 differentially expressed human homologous CREB targets in common with pediatric AML. Among them, we demonstrated that CREB overexpression increased CCAAT-enhancer-binding protein-δ (C/EBPδ) levels to cause myeloid differentiation arrest, and the silencing of CREB-C/EBPδ axis restored myeloid terminal differentiation. Then, C/EBPδ overexpression was found to identify a subset of pediatric AML affected by a block of myeloid differentiation at monocytic stage who presented a significant higher relapse risk and the enrichment of aggressive signatures. Finally, this study unveils the aberrant activation of CREB-C/EBPδ axis concurring to AML onset by disrupting the myeloid cell differentiation process. We provide a novel in vivo model to perform high-throughput drug screening for AML cure improvement.

ICER expression inhibits leukemia phenotype and controls tumor progression.

The inducible cyclic AMP (cAMP) early repressor (ICER) and cAMP response element-binding protein (CREB) are transcriptional regulators of the cAMP-mediated signaling pathway. CREB has been demonstrated to be upregulated in the majority of childhood leukemias contributing to disease progression, whereas ICER, its endogenous repressor, was found to be downregulated. Our research focus has been the function of restored ICER expression. ICER exogenously expressed in cell lines decreases CREB protein level and induces a lowered clonogenic potential in vitro. It decreases the ability of HL60 to invade the extramedullary sites and to promote bone marrow angiogenesis in nonobese diabetic-severe combined immunodeficient mice, demonstrating its potential effects on tumor progression. ICER represses the majority of 96 target genes upregulated by CREB. It binds CRE promoters and controls gene expression restoring the normal regulation of major cellular pathways. ICER is subjected to degradation through a constitutively active form of the extracellular signal-regulated protein kinase, which drives it to the proteasome. We propose that ICER is downregulated in HL60 to preserve CREB overexpression, which disrupts normal myelopoiesis and promotes blast proliferation. These findings define the function of ICER as a tumor suppressor in leukemia. Unbalanced CREB/ICER expression needs to be considered a pathogenetic feature in leukemogenesis. The molecular characterization of this pathway could be useful for novel therapeutic strategies.

Vascular involvement in diabetic subjects with ischemic foot ulcer: a new morphologic categorization of disease severity.

OBJECTIVES: Arteriographic lesions of diabetic subjects with critical limb ischemia (CLI) and ischemic foot ulcer were reviewed retrospectively, to provide new criteria for stratification of these patients on the basis of their vascular involvement. PATIENTS: In 417 consecutive CLI diabetic subjects with ischemic foot ulcer undergoing lower limb angiography, lesions were defined as stenosis or occlusion, localization, and length (<5 cm, 5-10 cm, >10 cm). In a subgroup of 389 subjects, foot arteries also were evaluated. Patients then were categorized into 7 classes of progressive vascular involvement based on angiographic findings. RESULTS: Of the 2893 found lesions (55% occlusions) 1% were in the iliac arteries, whereas 74% were in below-the-knee (BTK) arteries. Sixty-six % of all BTK lesions were occlusions, and 50% were occlusions >10 cm (p<0.001 vs proximal segments). Occlusions of all BTK were present in 28% of patients, although there was patency of at least one foot artery in 55% of patients. The morphologic Class 4 (two arteries occluded and multiple stenoses of tibial/peroneal and/or femoral/popliteal vessels) was the most common (36%). An inverse correlation between morphologic class and TcPO2 was observed (r=-0.187, p=0.003). CONCLUSIONS: In CLI diabetic subjects with ischemic foot ulcer, the vascular involvement is extremely diffuse and particularly severe in tibial arteries, with high prevalence of long occlusions. A new morphologic categorization of these patients is proposed.

Prevalence of NIDDM and impaired glucose tolerance in Italy: an OGTT-based population study.

To provide complete prevalence data on diabetes mellitus in Italy (diagnosed and undiagnosed), a population survey was performed in the Health District of Cremona, a representative area of the Po river (north Italy). The survey is characterised by particular attention being paid to methodology, non-responders being investigated for possible selection biases affecting diagnosed and undiagnosed diabetes prevalence estimations. Out of a population aged 44 years or older from three municipalities, a random sample of 3097 subjects was selected to undergo an oral glucose tolerance test. In addition, past medical history, clinical and laboratory data were collected. A total of 1797 subjects participated (58%), and information on known diabetes status was obtained for 2618 persons (85%), also including 826 interviewed non-participating subjects. Overall rates were age-standardised according to the 1991 Italian census. Overall prevalence and 95% confidence interval of diagnosed diabetes was 8.5% (6.9-10.1) in males and 7.9% (6.7-9.3) in females over the age of 44 years; previously undiagnosed diabetes was 2.5% (1.4-3.6) in males and 3.4% (2.1-4.7) in females; glucose intolerance was 7.7% (5.7-9-7) in males and 8.9% (7.0-10.8) in females. Total diabetes prevalence above age 44 years, developed-world age, and sex standardised, was 10.7%. This study provides the first reliable prevalence estimation of impaired glucose tolerance, diagnosed and undiagnosed diabetes in Italy, according to World Health Organization criteria, and one of the few figures for Southern Europe. The role of body mass index on both prevalence of diabetes and cluster of cardiovascular risk factors is considered, with implications for prevention.

An Italian population-based study of the prevalence of diabetes: some methodological aspects.

The aim of the present study, which is part of a larger population survey, is to provide an Italian estimate of the total prevalence of diabetes taking into account the fraction of known diabetes among the non-responders. The study consists of a population survey. Out of the population aged over 40 ys. of two municipalities, Casalbuttano and Vescovato, in the Health District of Cremona, a random sample of 2,229 subjects was selected to undergo an OGTT. In addition, a series of anamnestic, clinical, and laboratory data were collected for each individual. Overall response rate was 63%. Prevalence of known diabetes was 7.8% (6.5%-9.1%, 95% CI), (F = 7.9%, M = 7.6%). The comparison between responders and the telephone-interviewed non-responders showed an unpredictable and age- and sex-dependently varying frequency of known diabetes, even if the overall differences were not statistically significant. This suggests that, to avoid biases, many efforts should be spent to collect information about diabetes also for non-responders. Prevalence of unknown diabetes was 3.2% (2.2%-4.2%, 95% CI), (F = 4.0%, M = 1.8%) and total diabetes was 10.9% (9.3%-12.5%, 95% CI) (F = 11.9%, M = 9.4%). In contrast to other countries, the relationship between unknown and known diabetes was less than 1 to 2, and the proportion of unknown diabetes was almost negligible below the age of 70. A positive family history and a lower level of triglycerides were associated with awareness of the disease (p = 0.0005 and p = 0.006, respectively), thus suggesting different behaviour towards one's own health between known and unknown diabetic subjects.