Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers

The lipid-layer technique allows reconstituting transmembrane proteins at a high density in microns size planar membranes and suspended to a lipid monolayer at the air/water interface. In this paper, we transferred these membranes onto two hydrophobic substrates for further structural analysis of reconstituted proteins by Atomic Force Microscopy (AFM). We used a mica sheet covered by a lipid monolayer or a sheet of highly oriented pyrolytic graphite (HOPG) to trap the lipid monolayer at the interface and the suspended membranes. In both cases, we succeeded in the transfer of large membrane patches containing densely packed or 2D-crystallized proteins. As a proof of concept, we transferred and imaged the soluble Shiga toxin bound to its lipid ligand and the ATP-binding cassette (ABC) transporter BmrA reconstituted into a planar bilayer. AFM imaging with a lateral resolution in the nanometer range was achieved. Potential applications of this technique in structural biology and nanobiotechnology are discussed.     

Circulating gammadelta T cells in young/adult and old patients with cutaneous primary melanoma

BACKGROUND: In a previous study we demonstrated the existence of numerical and functional alterations of gammadelta T cells in healthy elderly. Recently, we analysed the involvement of gammadelta T lymphocytes in malignant melanoma, describing a lower frequency of circulating gammadelta T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients. METHODS: In this study we investigated the existence of numerical and functional alterations of circulating gammadelta T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects. RESULTS: We demonstrated that the number of circulating gammadelta+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vdelta2 T cells whereas the number of Vdelta1 T cells was not affected. A higher percentage of gammadelta+ T cells producing TNF-alpha was found in old healthy donors, whereas a reduced number of TNF-alpha-producing gammadelta+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-gamma production. After a 10-day in vitro culture, both the percentage and the expansion index of gammadelta T cells, and in particular of Vdelta2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects. CONCLUSIONS: Our study demonstrates that the numerical and functional impairment of gammadelta T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of gammadelta T cells found in old healthy subjects. We suggest that a similar impairment of gammadelta T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma.     

Folk Phytotherapy in Paediatric Health Care in Central and Southern Italy: a Review

Complementary and alternative medicine (CAM) consists of systems and practices used to prevent or treat illness, and/or promote health and wellbeing, generally with herbal remedies. In recent decades, ethnobotanical and ethnopharmacological surveys have received increased attention among populations in central and southern Italy traditionally highly conservative regarding knowledge of folk medicine. This review highlights the use of wild and cultivated plants in paediatric health care. From a search of articles published between 1978 and 2017 we found a total of 34 containing reports of medicinal plants specifically used for treating children’s diseases, listing 83 taxa belonging to 37 families in 116 recorded cases of medicinal plant use. Tuscany has the highest number of plant use reports and the highest number of species used in a single region, followed by Campania and Sicily. Allium sativum is the most widely reported species, cited in eight regions. We identified eight ailment categories. Plants used as anthelmintics and for gastro-intestinal and skin diseases are widely used.     

Independent regulation of Rap1 and mitogen-activated protein kinase by the alpha chain of Go

Receptors coupled to G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) cascade. The intracellular pathways linking the alpha chains of these G proteins to MAPK activation are not completely understood. One of the signaling molecules which has been suggested to act downstream of Galpha(i/o) is the small G protein Rap1. We investigated the role of Rap1 in MAPK stimulation by Galpha(o) in Chinese hamster ovary (CHO) cells. Our previous results have shown that in this cell system activated Galpha(o) strongly potentiates the MAPK response to the epidermal growth factor (EGF) receptor. Rap1 regulation was examined in cells transfected with Rap1 and wild-type Galpha(o) or the activated mutant Galpha(o)-Q205L. Immunocytochemical analysis detected both Rap1 and the Galpha(o) subunit at the plasma membrane as well as on perinuclear cytoplasmic vesicles. Expression of wild-type Galpha(o) had no significant effect on the levels of activated Rap1. In contrast, Galpha(o)-Q205L virtually abolished the activation of Rap1 induced by EGF. Further experiments showed that MAPK stimulation by EGF was greatly inhibited by expression of activated Rap1, suggesting that Rap1 inhibition could mediate the effect of Galpha(o) on the MAPK cascade. However, Galpha(o)-Q205L efficiently inhibited the activation of Rap1 induced by fibroblast growth factor (FGF). We have previously found that the ability of FGF to activate MAPK is not modified by Galpha(o). In addition, expression of the GAP protein RAP1GAPII blocked Rap1 activation without affecting EGF- or FGF-dependent MAPK stimulation. These findings provide evidence for independent regulation of Rap1 and MAPK by the G(o )alpha chain.     

Mitochondrial non-syndromic sensorineural hearing loss: a clinical, audiological and pathological study from Italy, and revision of the literature

Over the last decade, a number of distinct mutations in the mtDNA (mitochondrial DNA) have been found to be associated with both syndromic and non-syndromic forms of hearing impairment. Their real incidence as a cause of deafness is poorly understood and generally underestimated. Among the known mtDNA mutations, the A1555G mutation in the 12S gene has been identified to be one of the most common genetic cause of deafness, and it has been described to be both associated to non-syndromic progressive SNHL (sensorineural hearing loss) and to aminoglycoside-induced SNHL. In the present study, we have investigated the presence of mtDNA alterations in patients affected by idiopathic non-syndromic SNHL, both familiar and sporadic, in order to evaluate the frequency of mtDNA alterations as a cause of deafness and to describe the audiological manifestations of mitochondrial non-syndromic SNHL. In agreement with previous studies, we found the A1555G mutation to be responsible for a relevant percentage (5.4%) of cases affected with isolated idiopathic sensorineural hearing impairment.     

Prognostic Role of Ventricular Ectopic Beats in Systemic Sclerosis: A Prospective Cohort Study Shows ECG Indexes Predicting the Worse Outcome

BackgroundArrhythmias are frequent in Systemic Sclerosis (SSc) and portend a bad prognosis, accounting alone for 6% of total deaths. Many of these patients die suddenly, thus prevention and intensified risk-stratification represent unmet medical needs. The major goal of this study was the definition of ECG indexes of poor prognosis.MethodsWe performed a prospective cohort study to define the role of 24h-ECG-Holter as an additional risk-stratification technique in the identification of SSc-patients at high risk of life-threatening arrhythmias and sudden cardiac death (SCD). One-hundred SSc-patients with symptoms and/or signs suggestive of cardiac involvement underwent 24h-ECG-Holter. The primary end-point was a composite of SCD or need for implantable cardioverter defibrillator (ICD).ResultsFifty-six patients (56%) had 24h-ECG-Holter abnormalities and 24(24%) presented frequent ventricular ectopic beats (VEBs). The number of VEBs correlated with high-sensitive cardiac troponin T (hs-cTnT) levels and inversely correlated with left-ventricular ejection fraction (LV-EF) on echocardiography. During a mean follow-up of 23.1±16.0 months, 5 patients died suddenly and two required ICD-implantation. The 7 patients who met the composite end-point had a higher number of VEBs, higher levels of hs-cTnT and NT-proBNP and lower LV-EF (p = 0.001 for all correlations). All these 7 patients had frequent VEBs, while LV-EF was not reduced in all and its range was wide. At ROC curve, VEBs>1190/24h showed 100% of sensitivity and 83% of specificity to predict the primary end-point (AUROC = 0.92,p<0.0001). Patients with VEBS>1190/24h had lower LV-EF and higher hs-cTnT levels and, at multivariate analysis, the presence of increased hs-cTnT and of right bundle branch block on ECG emerged as independent predictors of VEBs>1190/24h. None of demographic or disease-related characteristics emerged as predictors of poor outcome.ConclusionsVEBS>1190/24h identify patients at high risk of life-threatening arrhythmic complications. Thus, 24h-ECG-Holter should be considered a useful additional risk-stratification test to select SSc-patients at high-risk of SCD, in whom an ICD-implantation could represent a potential life-saving intervention.     

The Association between Overweight and Illegal Drug Consumption in Adolescents: Is There an Underlying Influence of the Sociocultural Environment?

Background

The aims of the study were to: a) Examine the distribution of gender-stratified body mass index (BMI), eating attitudes and use of addictive substances, under the hypothesis of a confluent prevalence of weight abnormalities, eating disorders and substance abuse. b) Demonstrate the extent to which family, peer-related and psychosocial factors are common elements in categories of compulsive behaviour.

Methodology/Principal Findings

In the present cross-sectional study, data were collected through self reported questionnaires administered to a large sample of 33,185 15–19 years old adolescents (ESPAD®Italia), divided into weight categories based on the BMI percentile distribution. Multinomial analyses were adopted to address the influence of social, family, leisure time factors, Eating Attitude Test (EAT26) on the association between weight categories and drug use.Recent drugs use was more frequent in overweight and underweight adolescents (p<0.05), especially in females. An EAT26 score ≥20 was more common in overweight adolescents. Multinomial analysis abolished the relationship between overweight and the use of most drugs, implicating self-esteem, parents'' educational level, and friendships as mediators of the association. Within the overweight category, adolescents reporting recent drug use, showed greater frequency of having drug-abusing friends (∼80%), and severe problems with parents and school (∼30%) compared to overweight adolescents without recent drug use.

Conclusion

The frequent association of overweight and substance use and the presence of common underlying social factors, highlights the need for an interdisciplinary approach involving individual-focused treatment models as well as public health, social and environmental changes to reduce food- and substances-related problems.     

Maternal Hypermethioninemia Affects Neurons Number,Neurotrophins Levels,Energy Metabolism,and Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Expression/Content in Brain of Rat Offspring

In the current study, we verified the effects of maternal hypermethioninemia on the number of neurons, apoptosis, nerve growth factor, and brain-derived neurotrophic factor levels, energy metabolism parameters (succinate dehydrogenase, complex II, and cytochrome c oxidase), expression and immunocontent of Na⁺,K⁺-ATPase, edema formation, inflammatory markers (tumor necrosis factor-alpha and interleukin-6), and mitochondrial hydrogen peroxide levels in the encephalon from the offspring. Pregnant Wistar rats were divided into two groups: the first one received saline (control) and the second group received 2.68 μmol methionine/g body weight by subcutaneous injections twice a day during gestation (approximately 21 days). After parturition, pups were killed at the 21st day of life for removal of encephalon. Neuronal staining (anti-NeuN) revealed a reduction in number of neurons, which was associated to decreased nerve growth factor and brain-derived neurotrophic factor levels. Maternal hypermethioninemia also reduced succinate dehydrogenase and complex II activities and increased expression and immunocontent of Na⁺,K⁺-ATPase alpha subunits. These results indicate that maternal hypermethioninemia may be a predisposing factor for damage to the brain during the intrauterine life.     

Co-culture of osteoblasts with immature dural cells causes an increased rate and degree of osteoblast differentiation

For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts.Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone.To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects.