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Minihepcidins are hepcidin agonists that have been previously shown to reverse iron overload and improve erythropoiesis in mice affected by non-transfusion-dependent thalassemia. Given the extreme anemia that occurred with the previous model of transfusion-dependent thalassemia, that model was inadequate for investigating whether minihepcidins can improve red blood cell quality, lifespan and ineffective erythropoiesis. To overcome this limitation, we generated a new murine model of transfusion-dependent thalassemia with severe anemia and splenomegaly, but sufficient red cells and hemoglobin production to test the effect of minihepcidins. Furthermore, this new model demonstrates cardiac iron overload for the first time. In the absence of transfusions, minihepcidins improved red blood cell morphology and lifespan as well as ineffective erythropoiesis. Administration of a minihepcidin in combination with chronic red blood cell transfusion further improved the ineffective erythropoiesis and splenomegaly and reversed cardiac iron overload. These studies indicate that drugs such as minihepcidins have therapeutic potential for patients with transfusion-dependent thalassemia., (Copyright© 2020 Ferrata Storti Foundation.)

In order to contribute to the elucidation of the biological role of carotenoids, the cellular response to hydrogen peroxide was analyzed in the red yeast R. mucilaginosa . For that, the wild strain C2.5t1, that produces β-carotene, torulene, and torularhodin, and the albino mutant 200A6 that is incapable of producing detectable amounts of these carotenoids, were grown in the presence of increasing concentrations of hydrogen peroxide. In spite of the difference in carotenoid content, the two strains presented comparable resistance to the pro-oxidant that showed a minimum inhibitory concentration of 6 mM. When subject to 1 h treatment with 16 mM hydrogen peroxide the two strains increased catalase but not superoxide activity, suggesting that catalase plays a major role in cell protection in both the wild strain and the albino mutant. Moreover, C2.5t1 reduced its carotenoid content by about 40% upon hydrogen peroxide treatment. This reduction in carotenoids was in agreement with a significant decrease of the transcript levels of genes involved in carotenoid biosynthesis. Since an excess of β-carotene may enhance reactive oxygen species toxicity, these results suggest that C2.5t1 modulates carotenoid content to counteract the pro-oxidant effect of hydrogen peroxide.

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with high heritability, although their underlying genetic factors are still largely unknown. Here we present a comprehensive genetic characterization of two ASD siblings from Sardinia by genome-wide copy number variation analysis and whole exome sequencing (WES), to identify novel genetic alterations associated with this disorder. Single nucleotide polymorphism (SNP) array data revealed a rare microdeletion involving CAPG , ELMOD3, and SH2D6 genes, in both siblings. CAPG encodes for a postsynaptic density (PSD) protein known to regulate spine morphogenesis and synaptic formation. The reduced CAPG mRNA and protein expression levels in ASD patients, in the presence of hemizygosity or a particular genetic and/or epigenetic background, highlighted the functional relevance of CAPG as a candidate gene for ASD. WES analysis led to the identification in both affected siblings of a rare frameshift mutation in VDAC3 , a gene intolerant to loss of function mutation, encoding for a voltage-dependent anion channel localized on PSD. Moreover, four missense damaging variants were identified in genes intolerant to loss of function variation encoding for PSD proteins: PLXNA2 , KCTD16 , ARHGAP21, and SLC4A1 . This study identifies CAPG and VDAC3 as candidate genes and provides additional support for genes encoding PSD proteins in ASD susceptibility.

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with β-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has β-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.

We describe a 3-year-old boy who, at age of 8 months, during investigations for upper respiratory tract infection was found to have an incidental grossly elevated CK of 20,000 UI/l. Investigations showed only mild calf hypertrophy and absent Gower's sign, normal cognitive function. Electromyography (EMG) showed myopathic features. Electrocardiography and echocardiography were normal. His muscle biopsy revealed myopathic features indicating Duchenne-type dystrophy. Immunohistochemistry for dystrophin N-terminal, C-terminal and mid-rod antibodies analysis showed the complete absence of dystrophin in the muscle fibers. Genetic studies showed a 141.1 Kb deletion removing muscle promoter, muscle exon 1, Purkinje promoter, Purkinje exon 1, dystrophin muscle enhancers similar to one previously reported in a DMD patient who exhibited some residual expression of dystrophin. The difference in dystrophin expression between these two patients might be due to the extension of deletions. The precise delimitation of the macrodeletion here described provides a better understanding of functional organization of the 5' end of the DMD gene., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Providing antiretroviral therapy for the HIV-infected population is a complex and challenging task. Treatment is often complicated by the shifting demographic of HIV-infected patients that now includes a large aging population in which patients often have multiple comorbidities. HIV clinicans are challenged with choosing the optimal combination of antiretrovirals based on potency, tolerability, bioavailability, and ease of administration. The issue of bioavailability is of paramount importance for those patients who can't swallow tablets, are unable to take anything by mouth before a procedure, or who need medication through a nasogastric tube or percutaneous endoscopic gastrostomy tube. A thorough search of several drug databases, a literature search of MEDLINE through Ovid, and a review of full prescribing information for each currently available antiretroviral drug, was performed to obtain insight into the bioavailability of antiretrovirals. Implications for the findings are discussed as they relate to adherence, resistance, alternative methods of administration, and the sometimes conflicting information on bioavailability that exists for various antiretroviral agents.

Background: The aim of this work has been to study the association between plasma asymmetric dimethylarginine (ADMA) concentrations and carotid stenosis in a group of 64 patients undergoing carotid endarterectomy (CEA)., Methods: Arginine, ADMA and symmetric dimethylarginine (SDMA) were measured using capillary electrophoresis with UV detection. An evaluation of plasma concentrations of total cysteine (tCys) and total homocysteine (tHcy) was also performed., Results: Pearson's analysis show a positive correlation between ADMA and carotid stenosis (r=0.37, p=0.003), which is also confirmed after stepwise multiple linear regression analysis. ADMA plasma concentrations were significantly associated with tHcy (r=0.40, p=0.001) and to a lesser extent, even if not significantly, with tCys (r=0.23, p=0.07)., Conclusions: Our data suggest that plasma ADMA is involved in carotid narrowing after CEA intervention. This suggests that this molecule may have an important role in the events that lead to stenosis.

Even though the incidence of anal cancer among men who have sex with men (MSM) is higher than the incidence of cervical cancer among women, few MSM are identified as high-risk patients in primary care or have received vaccination for human papillomavirus (HPV), the most common sexually transmitted infection worldwide, with 6.2 million new infections each year. The authors review the current literature on diagnosis and basic management of genital, oral, and anal HPV infection. Early diagnosis and treatment of patients with HPV infection is important because this infection causes patients substantial distress even in its benign manifestations. It has also been implicated in a host of cancers, including oral, cervical, penile, and anal cancers and is an independent risk factor for the development of human immunodeficiency virus infection. The incidence of HPV infection drops in women older than 30 years but remains high for MSM in all age ranges. For all of these reasons, physicians should routinely assess the sexual practices of all male patients, especially MSM, and educate them on the HPV infection risks, diagnosis, and treatment options. Physicians can have a significant impact in the primary prevention of HPV by routinely offering HPV vaccination to male patients younger than 26 years.

The role of neurotransmitter amino acids (NAAs) in the functioning of the nervous system has been the focus of increasingly intense research over the past several years. Among the various amino acids that have important roles as neurotransmitters, there are alanine (Ala), glutamic acid (Glu), aspartic acid (Asp), serine (Ser), taurine (Tau) and glycine (Gly). NAAs are present in plasma, cells and--at trace levels--in all biological fluids, but complex components in biological matrices make it difficult to determine them in biological samples. We describe a new capillary electrophoresis (CE) method with laser-induced fluorescence detection by which analytes are resolved in less than 12 minutes in a 18 mmol/L phosphate run buffer at pH 11.6. The use of elevated temperatures during sample derivatization leads to a drastic reduction in the reaction time, down to 20 min, compared to the 6-14 h usually described for reactions between FITC and amino acids at room temperature. In order to demonstrate its wide range of applications, the method was applied to the analysis of NAA in human plasma and in other sample types, such as red blood cells, urine, cultured cells, cerebrospinal fluid, saliva and vitreous humor, thus avoiding the typical limitations of other methods, which are normally suitable for use with only one or two matrix types.

In this work we describe a new method for taurine quantification in plasma by capillary electrophoresis laser-induced fluorescence detection. Taurine is derivatized with fluorescein isothiocyanate at 100 degrees C in 20 min. These conditions allow to reduce the pre-analytical times and to derivatize quantitatively the taurine contained in the reaction mixture, contrary to the room temperature derivatization commonly adopted. FITC-taurine adduct is analyzed in an uncoated fused-silica capillary, 75 mum ID and 40 cm effective length using a 20 mmol/L tribasic sodium phosphate buffer pH 11.8, at 22 kV. To avoid the typical problems due to instability of FITC-adduct, we use the homocysteic acid as internal standard. The loss of FITC-taurine signal during the sequence analysis is compensated by the same loss of FITC-internal standard adduct, thus giving a noteworthy improvement in the assay precision. The method shows a good reproducibility of the migration times (coefficient of variation, CV%, 1.93) and the peak areas (CV%, 3.65). Intra- and interassay CV were 4.63 and 6.44%, respectively, and analytical recovery was between 98.1 and 102.3%. Assay application was tested measuring taurine plasma levels in 50 healthy volunteers in which a mean value of 60.2 +/- 17.9 micromol/L was found. Moreover, the applicability of the method was also checked on energy drinks and milk.

Protein modification due to S-glutathio(ny)lation, usually a reversible process in intact cells, arises interest as a possible mode of regulatory events that may potentially modify a large number of cellular processes. However, since less than 1% of the total protein is S-thiolated in resting cells, high sensitivity methods are required for its evaluation. We set up a new method by CE with LIF detection that allows to measure all forms of intracellular GSH involved in the process. For total and reduced glutathione, cell lysates were rapidly derivatized by 5-iodoacetoamidofluorescein (5-IAF), a selective reagent which traps thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 47 cmx75 microm id capillary by using 7 mmol/L sodium phosphate at pH 11.6. For the evaluation of S-glutathio(ny)lation, intracellular proteins from cell lysates were precipitated and washed to eliminate free GSH. After protein resuspension with NaOH and reduction treatment with tri-n-butylphosphine (TBP), the freed GSH was dried in a vacuum concentrator and directly dissolved in the derivatization mixture. GSH-IAF adduct was detected in a 6 mmol/L sodium phosphate, 3 mmol/L boric acid, and 75 mmol/L N-methylglucamine run buffer in less than 5 min. The high sensitivity ensured by 5-IAF use and sample concentration, allowed to quantify GSH at levels as low as 5 nmol/L, value suitable for the evaluation of protein S-glutathio(ny)lation. The method suitability was checked both in HUVEC and ECV304 cultured cells.

Methionine is an important amino acid involved in protein synthesis and transmethylation reactions. It is also the precursor of homocysteine and cysteine, two important risk factors for cardiovascular diseases. As homocysteine research has gained impulsion, the evaluation of plasma methionine concentrations has acquired importance. Methionine measurement generally has been performed by HPLC after o-phthalaldehyde derivatization. Its separation from other amino acids is time-consuming. We set up a new specific capillary electrophoresis method in which analyte derivatization was avoided by sample concentration before analysis. Methionine was detected by UV absorbance at 204 nm with a detection limit of 0.5 micromol/L. By a capillary with an effective length of 50 cm filled with 125 mmol/L Tris phosphate buffer at pH 2.3, the separation occurred in less than 14 min. Precision tests indicated a good test repeatability for both migration times (coefficient of variation [CV]<0.3%) and areas (CV<2.0%). Moreover, a good reproducibility of intraassay and interassay tests was obtained (CV<2.9% and CV<3.5%, respectively). The Passing-Bablok regression and the Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference HPLC assay are similar. Assay performance was evaluated measuring methionine concentrations in retinal venous occlusive disease.

Guanidinoacetic acid (GAA) measurement has recently become of great interest for the diagnosis of creatine (Cn) metabolism disorders, and research calls for rapid and inexpensive methods for its detection in plasma and urine in order to assess a large number of patients. We propose a new assay for the measurement of GAA by a simple CZE UV-detection without previous sample derivatization. Plasma samples were filtered by Microcon-10 microconcentrators and directly injected into the capillary, while for urine specimens a simple water dilution before injection was needed. A baseline separation was obtained in less than 8 min using a 60.2 cm x 75 microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer pH 2.25 at 15 degrees C. The performance of the developed method was assessed by measuring plasma creatinine and Cn in 32 normal subjects and comparing the data obtained by the new method with those found with the previous CE assay. Our new method seems to be an inexpensive, fast and specific tool to assess a large number of patients both in clinical and in research laboratories.

Traditional clinical assays for nonprotein nitrogen compounds, such as creatine and creatinine, have focused on the use of enzymes or chemical reactions that allow measurement of each analyte separately. Most of these assays are mainly directed to urine quantification, so that their applicability on plasma samples is frequently hard to perform. This work describes a simple free zone capillary electrophoresis method for the simultaneous measurement of creatinine and creatine in human plasma. The effect of analytical parameters such as concentration and pH of Tris-phosphate running buffer and cartridge temperature on resolution, migration times, peak areas, and efficiency was investigated. Good separation was achieved using a 60.2-cm x 75-microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer, pH 2.25, at 15 degrees C, in less than 8 min. We compared the present method to a validated capillary electrophoresis assay, by measuring plasma creatinine in 120 normal subjects. The obtained data were compared by the Passing-Bablok regression and the Bland-Altman test. Moreover the performance of the developed method was assessed by measuring creatine and creatinine in 16 volunteers prior to and after a moderate physical exercise.

Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4 degrees C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5-iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 57 cm x 75 microm ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and gamma-glutamylcysteine (GluCys) were baseline-resolved in approximately 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro-oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers.

The aim of this work was to study the association between plasma thiol levels and percentage carotid narrowing in a group of 68 patients who underwent a carotid endarterectomy, pertained as a risk factor for vascular and cardiovascular disease. Total plasma thiols were measured by capillary electrophoresis laser-induced fluorescence. The mean values of the hematological parameters studied were within normal limits and 25% of the patients were hyperhomocysteinemic (homocysteine >15 micromol/L). Pearson's correlation between carotid narrowing degree and the most common risk factors for atherosclerosis showed a positive relationship only between carotid narrowing degree and cysteine levels (r=0.252; p<0.05). Stepwise multiple linear regression with carotid narrowing degree as the dependent variable, and cysteine, homocysteine, age, triglyceride and low-density lipoprotein-cholesterol as independent variables confirmed that cysteine was significantly associated with these variables. By regrouping the population according to cysteine and homocysteine concentration percentiles, we found positive correlation between these parameters and median values of carotid narrowing degree. Our study provides experimental evidence to confirm that plasma homocysteine and cysteine are involved in carotid narrowing after carotid endarterectomy intervention, suggesting that cysteine may be involved in the deleterious molecular mechanisms active in carotid stenosis.