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Pulmonary segmentectomy is a common surgical procedure in thoracic surgery nowadays. Though this technique helps preventing postoperative pulmonary function loss, potential challenges are the management of air leaks and the difficulty of palpating ground-glass components during surgery, as well as how to deal with the intersegmental planes. Several techniques have been proposed for the identification and treatment of the intersegmental planes during sublobar anatomical resections: this review focuses on preoperative planning and workup and intraoperative strategies. Three-dimensional computed tomography bronchography and angiography (3D-CTBA), virtual-assisted mapping (VAL-MAP) using bronchoscopy multi-spot dye marking and three-dimensional computed tomography (3D-CT) are preoperative tools that may facilitate the planning of operation. Inflation-deflation techniques, infrared-fluorescence-enhanced method combined with bronchial and intravenous injection of indocyanine green (ICG) and near-infrared fluorescence (NIF) mapping with ICG have been described as intraoperative strategies to identify the intersegmental plane. The treatment and section of the intersegmental planes is mainly accomplished by stapler and electrocautery or energy devices. The use of staplers reduces postoperative air leaks, bleeding risks and operative time but seems to reduce preserved lung volume, compromising adjacent lung expansion; in addition, higher costs and sometimes non-adequate oncological margins, being a non-anatomical technique have been described. The electrocautery and energy devices allow for a more anatomical and precise dissection maintaining safe oncological margins, with a better lung expansion and so an increased postoperative lung function. Time consuming procedure and frequent requirement of aero-haemostatic tools to treat air and blood leaks are the main drawbacks. In conclusion, there are several methods to identify and treat the intersegmental planes but there are no significant differences between the different tools, therefore the use of one technique rather than another depends overall on surgeon's preference and the location of the segment., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jtd.2020.01.09). The series “Early Stage Lung Cancer: Sublobar Resections are a Choice?” was commissioned by the editorial office without any funding or sponsorship. GM serves as an unpaid editorial board member of Journal of Thoracic Disease from Jan 2017 to Dec 2020. The other authors have no other conflicts of interest to declare., (2020 Journal of Thoracic Disease. All rights reserved.)

Eubacterium tarantellae was originally cultivated from the brain of fish affected by twirling movements. Here, we present the draft genome sequence of E. tarantellae DSM 3997, which consists of 3,982,316 bp. Most protein-coding genes in this strain are similar to genes of Clostridium bacteria, supporting the renaming of E. tarantellae as Clostridium tarantellae ., (Copyright © 2020 Bano et al.)

Context: There is little information on cognitive function in Silver-Russell syndrome (SRS), and no neuroimaging studies are available so far., Objective: To assess cognitive function and brain volumes in patients with SRS., Design/setting: Wechsler Intelligence Scale and brain magnetic resonance on a 3-Tesla scanner with Voxel-based morphometry analysis were performed between 2016 and 2018 in a single tertiary university center., Partecipants: 38 white subjects with clinical diagnosis of SRS confirmed by molecular analysis: 30 of these patients (mean age 12.6 ± 10 years) were enrolled for cognitive assessment; 23 of the 30 performed neuroimaging sequences. A control group of 33 school-aged children performed cognitive assessment while 65 age and sex-matched volunteers were included for the neuroradiological assessment., Main Outcomes: Intelligence quotient, Verbal Comprehension Index (VCI), Perceptual Reasoning Index (PRI), Working Memory Index (WMI), Processing Speed Index, and brain volume., Results: The mean overall IQ score was 87.2 ± 17, and it was significantly lower in the maternal uniparental disomy of chromosome 7 (mUPD7) group at the age of 6 to 16 years compared to loss of methylation on chromosome 11p15 (11p15 LOM) group and to controls. VCI, PRI, and WMI were significantly higher in 11p15 LOM group and in control group than in mUPD7 group at the age of 6 to 16 years. There were no significant differences in cognitive scores between 11p15 LOM school-aged patients and the control group. SRS patients showed lower brain volume compared to controls at the frontal/temporal poles and globi pallidi., Conclusions: Patients with mUPD7 had an impaired cognitive profile. The brain volume at the frontal/temporal lobes and at the globi pallidi was reduced in patients with SRS., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Obstructive sleep apnea (OSA) is a prevalent condition. OSA is defined as very severe when the apneas/ hypopneas index (AHI) is greater than 60 events/hour. However, its prevalence remains uncertain. The aim of this study was to describe the prevalence and characteristics from patients with OSA according to severity. We performed a retrospective analysis of patients > 18 years, assessed by respiratory polygraph. We evaluated a total of 5670 patients with a mean of age of 54.4 ± 14.3 years old, of which 3606 were included in the OSA group. The overall prevalence of very severe OSA was 4.8%, being higher in men than in women (6.8 vs 1.9%). This group of patients was younger than the less severe groups (54.7 ± 13.9: mild OSA; 56.5 ± 12.9: moderate OSA; 57.9 ± 13.0: severe OSA vs 51.5 ± 12.9 years in the very severe patients; p < 0.0001). The results showed a significant increase in the body mass index (BMI) of patients with very severe OSA (31.6 ± 6.9: mild OSA, 32.8 ± 6.6: moderate OSA, 34.0 ± 6.8 severe OSA vs 37.1 ± 8.5 extreme severity; p < 0.0001), with similar results in men, but not in women. Finally, drowsiness due to ESS > 10, showed a significant increase in the very severe group of both sexes; 43.2% vs 31.5% (severe OSA); 26.4% (moderate OSA) and 23.8% (mild OSA). Very severe OSA presented a prevalence of 4.8% of patients with OSA. According to previous publications in other studied populations, we found that patients with very severe OSA are younger and more obese than the less severe groups.

A rare case of a spontaneous coronary artery dissection (SCAD) in the postpartum of a 32-year-old woman is presented. Pregnancy-related SCAD is a potentially life-threatening condition. SCAD is triggered by nonatherosclerotic disease, typically affects young-aged women and is still often underdiagnosed. Cardiac magnetic resonance is crucial to demonstrate the ischemic origin of the disease. A conservative management is generally preferred in stable SCAD as most dissected segments will heal spontaneously over a few months. Additionally, the percutaneous coronary intervention is associated with high rate of complications and procedural failure. The aim of this case was to improve awareness of SCAD and to advise clinicians seeking aid with patient management., (© 2019 Wiley Periodicals, Inc.)

Excessive mitochondrial matrix Ca 2+ and oxidative stress leads to the opening of a high-conductance channel of the inner mitochondrial membrane referred to as the mitochondrial permeability transition pore (mtPTP). Because mtPTP opening can lead to cell death under diverse pathophysiological conditions, inhibitors of mtPTP are potential therapeutics for various human diseases. High throughput screening efforts led to the identification of a 3-carboxamide-5-phenol-isoxazole compounds as mtPTP inhibitors. While they showed nanomolar potency against mtPTP, they exhibited poor plasma stability, precluding their use in in vivo studies. Herein, we describe a series of structurally related analogues in which the core isoxazole was replaced with a triazole, which resulted in an improvement in plasma stability. These analogues were readily generated using the copper-catalyzed "click chemistry". One analogue, N-(5-chloro-2-methylphenyl)-1-(4-fluoro-3-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (TR001), was efficacious in a zebrafish model of muscular dystrophy that results from mtPTP dysfunction whereas the isoxazole isostere had minimal effect., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The yeast cell wall is composed of mannoproteins, β-1,3/β-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-β-1,3/anti-β-1,6-glucan antibodies for β-1,3/β-1,6-glucans. Binding specificity of these biomolecules were validated using penta- N -acetylchitopentaose, α-mannans, laminarin (short β-1,3-glucan chain) and gentiobiose (2 glucose units linked in β 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance x t , dissociate rate k off and the lowest force ( f eq ) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of β-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2019 The Author(s).)

This study reports biosynthesis of gold-nanoparticles (AuNPs) by using β-d-glucans isolated from the yeast Yarrowia lypolitica D1. β-d-glucans serve as reducing and stabilizing mediators that induce the formation of AuNPs on the outer surface of the own β-d-glucan. The systems were physicochemically characterized by ultraviolet visible (UV-Vis) spectroscopy, high-resolution transmission electron microscopy (HR-TEM), scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and dynamic light scattering (DLS) analyses. The results revealed the generation of AuNPs with quasi-spherical shape or large one dimension (1D) gold-nanostructures (AuNSs) depending on the HAuCl 4 concentration. A cytotoxic study was assessed in mouse splenocytes. Contrary to that expected, important cytotoxicity was found in all β-d-gluc+AuNPs systems by an oxidative stress increase. This study discusses the cytotoxic mechanism, suggesting that the resulting β-d-gluc+AuNPs systems may not be candidates for the formulation of immunostimulants or nanocarriers for biomedical applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43 C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

Background: Violence against women represents a violation of a fundamental human right and is a significant cause of death and disability worldwide. In developing countries, this issue is particularly dramatic and in sub-Saharan Africa were reached 65% of women reporting domestic violence., Objective: In this study, we assessed the burden and pattern of domestic violence registered at Beira Central Hospital, Mozambique from 2011 to 2015., Methods: We performed a descriptive analysis of data collected at the CHB Legal Medicine Service., Results: In five years, are recorded a total amount of 1,491 admissions for domestic violence of which 1307 were females. About 80% of all female cases are represented by the 11-40 age range and, in almost 90% the aggressor was the current or past partner. More than 75% were cases of repeated violence and in more than 60% there were minors attending the phenomenon., Conclusion: It is crucial to act immediately and with a multi-disciplinary approach in order to fight domestic violence, especially against women due to its dramatic consequences as isolation, inability to work, loss of wages, lack of participation in regular activities and limited ability to care for themselves and their children.

Introduction : Hypertension (HT) and obstructive sleep apnea-hypopnea syndrome (OSA) are prevalent and frequently related pathologies. The predictive value of screening questionnaires in cardiovascular settings is not totally clear and could be useful to select candidates for the sleep test. Materials and Methods : We performed a prospective study in adults with suspected HT referred to a day-care hospital. Epworth Sleepiness Scale (ESS), Berlin, and STOP-BANG (SBQ) questionnaire were checked against data from home-based respiratory polygraphy (RP). We calculated sensitivity (S) and specificity (Sp), positive and negative predictive values (PPV and NPV), and the area under the ROC curve (AUC-ROC) for each questionnaire and their combinations. Results : We analyzed 382 patients; 234 men (61.3%) and 148 women, mean age: 54.5 ± 13.7 years, body mass index (BMI): 33.1 ± 7.8 kg/m 2 , ESS: 7.4 ± 4.7. Seventy-eight percent had an apnea-hypopnea index (AHI) >5 events per hour (eV/h) and 58% presented >15 eV/h (mean: 17.5 ± 9.3 eV/h). With regard to clinically significant OSA (AHI > 15 eV/h), 5 SBQ components showed S: 100% (CI: 97-100) and Sp: 98% (CI: 95-99) with a NPV of 100, a PPV of 97.8, and an AUC-ROC of 0.90 ( p < 0.0001 ). ESS > 10 + high-risk Berlin did not perform as effectively. Conclusion : In a population of HT patients, SBQ > 5 performed better at identifying patients with >15 eV/hour with a high discrimination power.

Background: Several studies suggested the association between tobacco and cannabis smoking and the risk of primary spontaneous pneumothorax (PSP), but none demonstrated cannabinoids in human lung tissues., Objectives: The aim of this study was to identify cannabinoids in lung specimens of young cannabis smokers, operated for PSP, and investigate on their pathologic findings, to determine the role of cannabis in PSP pathogenesis., Method: A prospective, multicenter study was conducted, enrolling patients admitted for PSP. Inclusion criteria were PSP requiring surgical treatment and history of cannabis smoking, associated or not to tobacco. Control cases were nonsmokers, and tobacco only smokers operated for PSP. Lung apex wedge resection by video-assisted thoracic surgery was performed. Two lung specimens, for pathological and toxicological examination, were taken from each patient., Results: Twenty-nine male patients were enrolled: 21 (72.4%) tobacco and cannabis smokers, 2 (7%) cannabis only smokers, 3 (10.3%) tobacco only smokers, 3 (10.3%) nonsmokers; all underwent lung apicectomy, 4 bilateral surgery, for a total of 33 procedures. Typical PSP pathologic findings were mainly detected in control cases, other alterations in cannabis users. Lung specimens resulted positive for cannabinoids on 22/33 cases (19/22 reported being, 3/22 not being cannabis smokers), negative on 11/33 (3/11 reported not being, 7/11 having been cannabis smokers, 1/11 cannabis smoker)., Conclusions: Our study demonstrated the presence of cannabinoids and particular pathologic alterations in lung tissues of young cannabis smokers with PSP, supporting the correlation between this disease and marijuana abuse and suggesting spontaneous pneumothorax "secondary to marijuana" as a new nosological entity., (© 2019 S. Karger AG, Basel.)

Background: Pulmonary and mediastinal paragangliomas are rare tumors that may have neuroendocrine activity or be non-functional, incidental, in asymptomatic patients, or causing mass effect symptoms. Although being low-grade tumors, they can display an aggressive behaviour, developing local infiltration and distant metastases. We report our experience with three endothoracic paragangliomas and a Literature review, to point out diagnostic difficulties and problems related to surgical treatment., Methods: From 2009 to 2017, we treated 3 patients with histological diagnosis of paraganglioma: 2 pulmonary, 1 mediastinal. No one presented catecholamine-secreting syndromes; pulmonary cases were asymptomatic, while the mediastinal one had aspecific cough and dyspnea. Imaging diagnosis was based on chest computerized tomography (CT) and magnetic resonance imaging (MRI) scan. No patient had preoperative histological diagnosis. Intraoperative pathological examination was suggestive for malignancy: in pulmonary cases, wedge resection and lobectomy were performed; the middle mediastinal mass was completely removed after challenging dissection, isolation and section of numerous vascular pedicles., Results: Postoperative course was uneventful in all cases. No patient received adjuvant treatments. At a median follow-up of 47 months (range, 6-102 months), two patients are alive, without local or distant recurrence; one patient died 6 months after surgery, due to disease progression., Conclusions: Endothoracic paragangliomas, rare and often asymptomatic tumors, are of difficult diagnosis and should be considered malignant tumors, due to the potential aggressive behaviour of cases with high mitotic index and the frequent possibility of recurrence and metastases. Surgical resection is the treatment of choice and careful intraoperative manipulation is recommended, due to the high vascularity of these tumors, to prevent complications. After complete excision, long-term prognosis is generally good. However, even after surgical removal, a close, periodical and life-long follow-up is mandatory., Competing Interests: Conflicts of Interest: Presented on the 17th October 2017, as Poster, at the IASLC 18th World Conference on Lung Cancer, Yokohama, Japan, 15–18 October 2017.

Idebenone is a hydrophilic short-chain coenzyme (Co) Q analogue, which has been used as a potential bypass of defective complex I in both Leber Hereditary Optic Neuropathy and OPA1-dependent Dominant Optic Atrophy. Based on its potential antioxidant effects, it has also been tested in degenerative disorders such as Friedreich's ataxia, Huntington's and Alzheimer's diseases. Idebenone is rapidly modified but the biological effects of its metabolites have been characterized only partially. Here we have studied the effects of quinones generated during in vivo metabolism of idebenone with specific emphasis on 6-(9-carboxynonyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (QS10). QS10 partially restored respiration in cells deficient of complex I or of CoQ without inducing the mitochondrial permeability transition, a detrimental effect of idebenone that may offset its potential benefits [Giorgio et al. (2012) Biochim. Biophys. Acta 1817: 363-369]. Remarkably, respiration was largely rotenone-insensitive in complex I deficient cells and rotenone-sensitive in CoQ deficient cells. These findings indicate that, like idebenone, QS10 can provide a bypass to defective complex I; and that, unlike idebenone, QS10 can partially replace endogenous CoQ. In zebrafish (Danio rerio) treated with rotenone, QS10 was more effective than idebenone in allowing partial recovery of respiration (to 40% and 20% of the basal respiration of untreated embryos, respectively) and allowing zebrafish survival (80% surviving embryos at 60 h post-fertilization, a time point at which all rotenone-treated embryos otherwise died). We conclude that QS10 is potentially more active than idebenone in the treatment of diseases caused by complex I defects, and that it could also be used in CoQ deficiencies of genetic and acquired origin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Gynecomastia is a common finding in males, with an incidence that varies widely globally. In 10-25% of cases, it is caused by drugs. Its pathophysiologic mechanism includes exposure to exogenous estrogens and medications that cause hypogonadism, antiandrogenic effects and hyperprolactinemia. Gynecomastia is associated with exposure to antiretroviral therapy (ART), particularly efavirenz. Sometimes surgery may be required as treatment. We report a case of a 46-year-old man receiving ART presenting with a marked bilateral breast enlargement who underwent bilateral mastectomy as the only successful treatment in a low-income setting.

Introduction: To identify patients at risk for obstructive sleep apnea (OSA) syndrome at a specialized hypertension center, we administered questionnaires and used respiratory polygraphy (RP)., Results: We studied 168 patients (64.8% men and 35.2% women). Patients' body mass index (BMI) was 34.7 ± 6.79 and Epworth Sleepiness Scale (ESS) scores were 8.01 for male and 8.92 for women (p = 0.69). RP recordings revealed AHI (Apnea-Hypopnea Index) of 18.03 ± 15.7, an ODI (Oxygen Desaturation Index) of 18.6 ± 15.2, and a time oxygen saturation <90% (%) of 20.8 ± 24.3. Around 44% of patients had an AHI of >15 events/h, and continuous positive airway pressure (CPAP) was recommended to 69 patients (41.07%). Pulse wave velocity (PWV) showed high values in AHI > 15/h (p = 0.050), and carotid intima-media thickness (IMT) did not correlate with AHI > 15; right IMT: 0.83 ± 1.3 versus 0.78 ± 0.13 mm (p = 0.41) and 0.82 ± 0.16 versus 0.78 ± 0.19 mm (p = 0.40). However, we find correlation with carotid plaque (p = 0.046). The ACC/AHA calculator revealed a gradual increase in the risk of cardiovascular events: 8.7% with AHI < 5/h, and 30.3% in severe OSA., Conclusions: In hypertension (HT) patients, RP revealed a high prevalence of OSA associated with carotid artery disease, high PWV, and increased cardiovascular risk.

Duchenne muscular dystrophy (DMD) is a severe muscle disease of known etiology without effective, or generally applicable therapy. Mitochondria are affected by the disease in animal models but whether mitochondrial dysfunction is part of the pathogenesis in patients remains unclear. We show that primary cultures obtained from muscle biopsies of DMD patients display a decrease of the respiratory reserve, a consequence of inappropriate opening of the permeability transition pore (PTP). Treatment with the cyclophilin inhibitor alisporivir - a cyclosporin A derivative that desensitizes the PTP but does not inhibit calcineurin - largely restored the maximal respiratory capacity without affecting basal oxygen consumption in cells from patients, thus reinstating a normal respiratory reserve. Treatment with alisporivir, but not with cyclosporin A, led to a substantial recovery of respiratory function matching improved muscle ultrastructure and survival of sapje zebrafish, a severe model of DMD where muscle defects are close to those of DMD patients. Alisporivir was generally well tolerated in HCV patients and could be used for the treatment of DMD., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The yeast cell is surrounded by a cell wall conferring protection and resistance to environmental conditions that can be harmful. Identify the molecular cues (genes) which shape the biochemical composition and the nanomechanical properties of the cell wall and the links between these two parameters represent a major issue in the understanding of the biogenesis and the molecular assembly of this essential cellular structure, which may have consequences in diverse biotechnological applications. We addressed this question in two ways. Firstly, we compared the biochemical and biophysical properties using atomic force microscopy (AFM) methods of 4 industrial strains with the laboratory sequenced strain BY4743 and used transcriptome data of these strains to infer biological hypothesis about differences of these properties between strains. This comparative approach showed a 4-6-fold higher hydrophobicity of industrial strains that was correlated to higher expression of genes encoding adhesin and adhesin-like proteins and not to their higher mannans content. The second approach was to employ a multivariate statistical analysis to identify highly correlated variables among biochemical, biophysical and genes expression data. Accordingly, we found a tight association between hydrophobicity and adhesion events that positively correlated with a set of 22 genes in which the main enriched GO function was the sterol metabolic process. We also identified a strong association of β-1,3-glucans with contour length that corresponds to the extension of mannans chains upon pulling the mannosyl units with the lectin-coated AFM tips. This association was positively correlated with a group of 27 genes in which the seripauperin multigene family was highly documented and negatively connected with a set of 23 genes whose main GO biological process was sulfur assimilation/cysteine biosynthetic process. On the other hand, the elasticity modulus was found weakly associated with levels of β-1,6-glucans, and this biophysical variable was positively correlated with a set of genes implicated in microtubules polymerization, tubulin folding and mitotic organization.

Type 2 diabetes (T2D) is a disease characterized by impaired insulin secretion. The Wnt signaling transcription factor Tcf7l2 is to date the T2D-associated gene with the largest effect on disease susceptibility. However, the mechanisms by which TCF7L2 variants affect insulin release from β-cells are not yet fully understood. By taking advantage of a tcf7l2 zebrafish mutant line, we first show that these animals are characterized by hyperglycemia and impaired islet development. Moreover, we demonstrate that the zebrafish tcf7l2 gene is highly expressed in the exocrine pancreas, suggesting potential bystander effects on β-cell growth, differentiation and regeneration. Finally, we describe a peculiar vascular phenotype in tcf7l2 mutant larvae, characterized by significant reduction in the average number and diameter of pancreatic islet capillaries. Overall, the zebrafish Tcf7l2 mutant, characterized by hyperglycemia, pancreatic and vascular defects, and reduced regeneration proves to be a suitable model to study the mechanism of action and the pleiotropic effects of Tcf7l2, the most relevant T2D GWAS hit in human populations.

F-ATP synthases convert the electrochemical energy of the H + gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F-ATP synthases can also undergo a Ca 2+ -dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca 2+ binding site and the mechanism(s) through which Ca 2+ can transform the energy-conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro , in vivo and in silico studies we (i) pinpoint the "Ca 2+ -trigger site" of the PTP to the catalytic site of the F-ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca 2+ -ATP hydrolysis, confer resistance to Ca 2+ -induced, PTP-dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F-ATP synthase to a channel and of its role in cell death., (© 2017 The Authors.)

The hypothesis that mitochondrial dysfunction can be a general mechanism for cell death in muscle diseases is 40 years old. The key elements of the proposed pathogenetic sequence (cytosolic Ca 2+ overload followed by excess mitochondrial Ca 2+ uptake, functional and then structural damage of mitochondria, energy shortage, worsened elevation of cytosolic Ca 2+ levels, hypercontracture of muscle fibers, cell necrosis) have been confirmed in amazing detail by subsequent work in a variety of models. The explicit implication of the hypothesis was that it "may provide the basis for a more rational treatment for some conditions even before their primary causes are known" (Wrogemann and Pena, 1976, Lancet, 1, 672-674). This prediction is being fulfilled, and the potential of mitochondria as pharmacological targets in muscle diseases may soon become a reality, particularly through inhibition of the mitochondrial permeability transition pore and its regulator cyclophilin D., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The seventeenth century is a period of transition from religious views that are not authentic but dogmatic about demonic influences to the application of scientific and methodological criteria in science. During Enlightenment there was an approach heavily influenced by ethical issues. In this context, there is a rational recognition of the value of man free from the teleological type references. Mental illnesses are treated using scientific criteria. During the seventeenth century clinical interest is also extended to psychosis and not only to neurosis. There are several significant changes in the care of psychiatric patient, and healthcare institutions are improved and increased. Many behaviors are inspired by the values of philanthropy.

Unlabelled: A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucose-grown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3- to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells., Importance: Ethanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Gestational diabetes mellitus (GDM) is defined as a glucose intolerance that occurs for the first time or it is first identified during pregnancy. The GDM etiology is multifactorial. It has not completely been established yet and several known risk factors may contribute to its onset. To date, there are no shared guidelines on the management and follow-up, especially regarding the low-income countries. In this paper, we describe the state of art about epidemiology, physiopathology, diagnosis, and management of GDM. Moreover, we focus on the current state in low income countries trying to outline basis for further research.

Unlabelled: . Efficacy of splint with heel off-loaded in children with lower limbs plaster cast. Randomised open label trial., Introduction: Plasters cast are medical devices widely used in paediatric orthopaedics; however, few studies have addressed the occurrence and prevention of device-related complications., Aim: To evaluate whether positioning a custom made splint with heel off-loaded, would relieve pain, reduce care interventions, the materials to maintain heel unloading position and, at the same time, improve comfort and reduce pressure sores in children with lower limb plaster., Methods: Children enrolled in the study were randomised to splint, provided with a custom made splint positioned underneath the lower limb plaster after surgery; in the second group (control) the plaster position was maintained with cushions., Results: A total of 57 children were enrolled from November 2014 to August 2015, 29 in the treatment group (50.8%) and 28 in the control group (49.2%). Pain median levels were 4 (splint) vs. 5 (control) p=ns; mean number of care intervention was 2.1 (splint) vs. 5.2 (control) p<0.0005; materials used were 1.3 (splint) vs. 2.8 (control) p<0.0005; perceived comfort was 8.9 (splint) vs. 7.6 (control) p<0.001. A difference in the total number of pressure sores was observed with 1 case in the splint group vs. 5 cases in the control group RR 0.21 (IC 95% -1.88-2.29; P 0.079)., Conclusions: The use of splint is effective in reducing care intervention the use of other materials and in improving comfort, but not in relieving pain.

In this chapter, we are presenting methods to monitor and quantify in vivo canonical Wnt signaling activities at single-cell resolution in zebrafish. Our technology is based on artificial enhancers, obtained by polymerization of TCF binding elements, cloned upstream to ubiquitous or tissue-specific promoters. The different promoter/enhancer combinations are used to drive fluorescent protein reporter constructs integrated in the zebrafish germline by microinjection of fertilized zebrafish eggs. Fish with a single integration site are selected by Mendelian analysis of fluorescent carriers, and heterozygous offspring are used to monitor and quantify canonical Wnt activities. Open source public domain software such as ImageJ/Fiji is used to calculate the integrated densities in the region of interest and compare the effect of experimental conditions on control and treated animals.

The mitochondrial permeability transition pore (mtPTP) is a Ca(2+) -requiring mega-channel which, under pathological conditions, leads to the deregulated release of Ca(2+) and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5-(3-hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3-carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50 <0.39 μM) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC50 >100 μM). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N-(3-chloro-2-methylphenyl)-5-(4-fluoro-3-hydroxyphenyl)isoxazole-3-carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock., Importance: The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock., (Copyright © 2015 Ene et al.)

The Saccharomyces cerevisiae cell surface is endowed with some relevant technological properties, notably antimicrobial and biosorption activities. For these purposes, yeasts are usually processed and packaged in an 'autolysed/dried' formula, which may have some impacts on cell surface properties. In this report, we showed using a combination of biochemical, biophysical and molecular methods that the composition of the cell wall of two wine yeast strains was not altered by the autolysis process. In contrast, this process altered the nanomechanical properties as shown by a 2- to 4-fold increased surface roughness and to a higher adhesion to the atomic force microscope tips of the autolysed cells as compared to live yeast cells. Besides, we found that the two strains harboured differences in biomechanical properties that could be due in part to higher levels of mannan in one of them, and to the fact that the surface of this mannan-enriched strain is decorated with highly adhesive patches forming nanodomains. The presence of these nanodomains could be correlated with the upregulation of flocculin encoding FLO11 as well as to higher expression of few other genes encoding cell wall mannoproteins in this mannan-enriched strain as compared to the other strain., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Five dammarane-type triterpenoids, five pentacyclic triterpenoids (three of them carrying a carboxylic acid group), and two aromadendrane-type sesquiterpenoids were isolated from an Argentinian collection of the liverwort Lepidozia chordulifera. Compounds were characterized by comparison of their spectral data with those previously reported and tested in their ability to control bacterial growth, biofilm formation, bacterial Quorum Sensing process (QS), and elastase activity of Pseudomonas aeruginosa, as well as bacterial growth and biofilm formation of Staphylococcus aureus. The key role played by biofilm and elastase activity in bacterial virulence make them a potential target for the development of antibacterial agents. The aromadendrane-type sesquiterpenoid viridiflorol was the most potent biofilm formation inhibitor, producing 60% inhibition in P. aeruginosa and 40% in S. aureus at 50µg/ml. Ursolic and betulinic acids (two of the pentacyclic triterpenoids isolated) were able to reduce 96 and 92% the elastase activity of P. aeruginosa at 50µg/ml, respectively. Among the analyzed triterpenoids, those that carry a dammarane skeleton were the most potent inhibitors of the P. aeruginosa biofilm formation and were active against both P. aeruginosa and S. aureus. Subsequently, a computer-assisted study of the triterpenoid compounds was carried out for a better understanding of the structure-activity relationships., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Candida albicans is an opportunistic pathogen. It adheres to mammalian cells through a variety of adhesins that interact with host ligands. The spatial organization of these adhesins on the cellular interface is however poorly understood, mainly because of the lack of an instrument able to track single molecules on single cells. In this context, the atomic force microscope (AFM) makes it possible to analyze the force signature of single proteins on single cells. The present study is dedicated to the mapping of the adhesive properties of C. albicans cells. We observed that the adhesins at the cell surface were organized in nanodomains composed of free or aggregated mannoproteins. This was demonstrated by the use of functionalized AFM tips and synthetic amyloid forming/disrupting peptides. This direct visualization of amyloids nanodomains will help in understanding the virulence factors of C. albicans., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Atomic force microscopy (AFM) is a useful tool for studying the morphology or the nanomechanical and adhesive properties of live microorganisms under physiological conditions. However, to perform AFM imaging, living cells must be immobilized firmly enough to withstand the lateral forces exerted by the scanning tip, but without denaturing them. This protocol describes how to immobilize living cells, ranging from spores of bacteria to yeast cells, into polydimethylsiloxane (PDMS) stamps, with no chemical or physical denaturation. This protocol generates arrays of living cells, allowing statistically relevant measurements to be obtained from AFM measurements, which can increase the relevance of results. The first step of the protocol is to generate a microstructured silicon master, from which many microstructured PDMS stamps can be replicated. Living cells are finally assembled into the microstructures of these PDMS stamps using a convective and capillary assembly. The complete procedure can be performed in 1 week, although the first step is done only once, and thus repeats can be completed within 1 d.

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specifications, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A reliable method to determine cell wall polysaccharides composition in yeast is presented, which combines acid and enzymatic hydrolysis. Sulphuric acid treatment is used to determine mannans, whereas specific hydrolytic enzymes are employed in a two sequential steps to quantify chitin and the proportion of β-(1,3) and β-(1,6)-glucan in the total β-glucan of the cell wall. In the first step, chitin and β-(1,3)-glucan were hydrolysed into their corresponding monomers N-acetylglucosamine and glucose, respectively, by the combined action of a chitinase from Streptomyces griseus and a pure preparation of endo/exo-β-(1,3)-glucanase from Trichoderma species. This step was followed by addition of recombinant endo-β-(1,6)-glucanase from Trichoderma harzianum with β-glucosidase from Aspergillus niger to hydrolyse the remaining β-glucan. This latter component corresponded to a highly branched β-(1,6)-glucan that contained about 75-80% of linear β-(1,6)-glucose linked units as deduced from periodate oxidation. We validated this novel method by showing that the content of β-(1,3), β-(1,6)-glucan or chitin was dramatically decreased in yeast mutants defective in the biosynthesis of these cell wall components. Moreover, we found that heat shock at 42 °C in Saccharomyces cerevisiae and treatment of this yeast species and Candida albicans with the antifungal drug caspofungin resulted in 2- to 3-fold increase of chitin and in a reduction of β-(1,3)-glucan accompanied by an increase of β-(1,6)-glucan, whereas ethanol stress had apparently no effect on yeast cell wall composition., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRAS(G12D) during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRAS(G12D) expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRAS(G12D) line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRAS(G12D) showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFβ and Notch pathways. Increase in TGFβ, following KRAS(G12D) activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for describing in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs., (© 2014. Published by The Company of Biologists Ltd.)

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy., (©2013 AACR.)

Background: Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h., Results: We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2-3 μm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young's Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton., Conclusions: Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway.

Over the past 20 years, the yeast cell wall has been thoroughly investigated by genetic and biochemical methods, leading to remarkable advances in the understanding of its biogenesis and molecular architecture as well as to the mechanisms by which this organelle is remodeled in response to environmental stresses. Being a dynamic structure that constitutes the frontier between the cell interior and its immediate surroundings, imaging cell surface, measuring mechanical properties of cell wall or probing cell surface proteins for localization or interaction with external biomolecules are among the most burning questions that biologists wished to address in order to better understand the structure-function relationships of yeast cell wall in adhesion, flocculation, aggregation, biofilm formation, interaction with antifungal drugs or toxins, as well as response to environmental stresses, such as temperature changes, osmotic pressure, shearing stress, etc. The atomic force microscopy (AFM) is nowadays the most qualified and developed technique that offers the possibilities to address these questions since it allows working directly on living cells to explore and manipulate cell surface properties at nanometer resolution and to analyze cell wall proteins at the single molecule level. In this minireview, we will summarize the most recent contributions made by AFM in the analysis of the biomechanical and biochemical properties of the yeast cell wall and illustrate the power of this tool to unravel unexpected effects caused by environmental stresses and antifungal agents on the surface of living yeast cells.

Saccharomyces cerevisiae and Candida albicans are model yeasts for biotechnology and human health, respectively. We used atomic force microscopy (AFM) to explore the effects of caspofungin, an antifungal drug used in hospitals, on these two species. Our nanoscale investigation revealed similar, but also different, behaviors of the two yeasts in response to treatment with the drug. While administration of caspofungin induced deep cell wall remodeling in both yeast species, as evidenced by a dramatic increase in chitin and decrease in β-glucan content, changes in cell wall composition were more pronounced with C. albicans cells. Notably, the increase of chitin was proportional to the increase in the caspofungin dose. In addition, the Young modulus of the cell was three times lower for C. albicans cells than for S. cerevisiae cells and increased proportionally with the increase of chitin, suggesting differences in the molecular organization of the cell wall between the two yeast species. Also, at a low dose of caspofungin (i.e., 0.5× MIC), the cell surface of C. albicans exhibited a morphology that was reminiscent of cells expressing adhesion proteins. Interestingly, this morphology was lost at high doses of the drug (i.e., 4× MIC). However, the treatment of S. cerevisiae cells with high doses of caspofungin resulted in impairment of cytokinesis. Altogether, the use of AFM for investigating the effects of antifungal drugs is relevant in nanomedicine, as it should help in understanding their mechanisms of action on fungal cells, as well as unraveling unexpected effects on cell division and fungal adhesion.

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.

The role of apoptosis in the persistence of hepatitis C virus (HCV) infection is controversial. Moreover, conflicting data on the modulation of this process by HCV proteins have been provided. We evaluated the susceptibility of peripheral lymphocytes from patients with chronic hepatitis C to apoptosis both spontaneous and after incubation with a chimeric Cucumber mosaic virus (CMV) carrying 180 copies of the synthetic R9 mimotope obtained from more than 200 hypervariable region-1 sequences of HCV. Resting T lymphocytes were found to be sensitized to apoptosis as a result of chronic HCV infection. The plant virus-derived vector R9-CMV displayed a strong pro-apoptotic effect associated with activation of both caspase-8 and -9, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. A parallel R9-CMV-mediated activation of endoplasmic reticulum-stress was suggested by the significant induction of BiP/GRP78, GADD153 and caspase-12. These data contribute to define the complex HCV/host interaction, and open new prospects for developing a plant-derived antigen-presenting system to strengthen host defences against persistent pathogens.

Unlabelled: Effectiveness of the transparent sterile dressing vs standard to fix the peripheral venous catheter (PVC), on the incidence of phlebitis. A randomized controlled trial., Introduction: The type of dressing could contribute to the incidence of phlebitis, infiltration and accidental removals but the results of the studies are contrasting and samples are limited., Aim: To compare the effectiveness of a transparent polyurethane sterile dressing on the rate of phlebitis associated to peripheral venous catheter (PVC) vs a non sterile sticking plaster in use in current practice (standard dressing)., Design: Randomized controlled trial. Participants. 1061 PVCs (703 patients, adults and children) at a research orthopedic hospital in the north of Italy; 540 PVCs allocated to receive the sterile and 521 the standard dressing., Results: 96 PVCs were excluded for phlebitis, 48 (9.6%) in the sterile and 48 (10.1%) in the standard dressing group, RR 0.96 (95%CI 0.697 - 1.335). Accidental removal of the PVCs was more frequent with the sterile dressing (9.6% vs 6.3%) but the number of catheters removed without complications was larger in the standard dressing group (48.9% vs 54.9% P=0.0503). Eighty-five PVCs were replaced for detachment of the dressing (50, 9.2% sterile and 35, 6.7% standard dressing). The cheapest transparent sterile dressing costs 32 cents while the standard 9 cents., Conclusions: A sticking non sterile plasters is not influential on the rate of phlebitis and ensures an good fix of the PVC compared the transparent sterile dressing to of polyurethane film.

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.