InterMiG: international differences in the therapeutic approach to migraine patients in specialized headache centers.

BACKGROUND: There is currently a wide therapeutic arsenal for migraine patients, without a single first-line preventive drug and we choose the different available alternatives taking into account comorbidities, national guidelines, previous treatments and personal experiences. Our objective was to evaluate the differences in the use of migraine treatments between neurologists from different countries. METHODS: This is a multi-centre observational study carried out by neurologists from specialized headache units in seven countries, retrospective with consecutive inclusion of all patients presenting with a migraine diagnosis, over a period of three months. RESULTS: A total of 734 patients were recruited but only 600 were considered in the analysis in order to homogenize the patient cohorts from countries: 200 Spain (ES), 100 Italy (IT), 85 Russia (RUS), 80 Germany (DE), 60 Portugal (PT), 45 Poland (PL) and 30 Australia (AU). 85.4 % of patients were women with a mean age of 42.6 ± 11.8 years. Considering previous and current preventive treatment, the order of use was: antidepressants (69.3 %), antiepileptic drugs (54.7 %), beta-blockers and antihypertensive drugs (49.7 %), OnabotulinumtoxinA (44.0 %) and others (36.2 %). Statistically significant differences were found between all pharmacological classes: antidepressants were commonly used in all countries, with the exception of Poland (AU: 76.7 %, IT: 71.0 %, DE: 60.0 %, PL: 31.1 %, PT: 71.7 %, RUS: 70.6 %, ES: 78.5 %; p < 0.0001); antiepileptic drugs were more frequently prescribed in Portugal, Australia and Spain (AU: 73.3 %, IT: 40.0 %, DE: 37.5 %, PL: 48.9 %, PT: 85.0 %, RUS: 29.4 % and ES: 69.0 %; p < 0.0001); beta-blockers and antihypertensive drugs were frequently used in all countries except Italy (AU: 60.0 %, IT: 14.0 %, DE: 53.8 %, PL: 48.9 %, PT: 68.3 %, RUS: 49.4 % and ES: 59.0 %; p < 0.0001); BTX-A were predominately used in Spain, Italy and Australia (AU:56.7 %, IT:58.0 %, DE:20.0 %, PL: 42.2 %, PT: 26.7 %, RUS: 24.7 % and ES: 58.5 %; p < 0.0001) and others were most frequently used in Poland (AU: 0.0 %, IT: 19.0 %, DE: 42.5 %, PL: 95.6 %, PT: 31.7 %, RUS: 3.5 % and ES: 49.5 %; p < 0.0001). If only patients without comorbidities are considered (200/600), statistically differences between countries persist in all preventive treatments. CONCLUSIONS: There is heterogeneity in the choice of preventive treatment between different countries. Prospective comparative studies of the different oral and subcutaneous alternatives would help to create a global therapeutic algorithm that would guarantee the best option for our patients.

Evaluation of the concomitant use of oral preventive treatments and onabotulinumtoxinA in chronic migraine: the PREVENBOX study.

BACKGROUND AND PURPOSE: OnabotulinumtoxinA is an effective preventive treatment for chronic migraine (CM). In CM, in addition to a reduction in headache frequency, a decreased reliance on oral prophylactics is also indicative of treatment effectiveness. This study aimed to quantify the change in the use of oral prophylactics after treatment with onabotulinumtoxinA in patients with CM. METHODS: This was a retrospective, multicentric, cross-sectional study. Patients with CM (International Classification of Headache Disorders-3beta) that had been treated with onabotulinumtoxinA were enrolled consecutively. We collected parameters related to each patient's pre-treatment situation, as well as their current situation, focusing on frequency and intensity of migraine, number of oral prophylactics and the respective cycle of onabotulinumtoxinA. Univariate and logistic regression analyses were performed. RESULTS: We included 542 patients, 90.0% of whom were taking oral preventive treatments. During treatment with onabotulinumtoxinA, 47.8% withdrew at least one prophylactic and 41.6% stopped using oral prophylactics altogether. Factors associated with a reduction or cessation of oral prophylactics were >50% improvement in frequency and intensity, remission to episodic migraine, use of topiramate as an initial treatment, increased number of infiltrations and shorter chronification period (P < 0.05). The multivariate analysis showed that a chronification period <20 months, more than five cycles of onabotulinumtoxinA, >50% improvement in pain intensity and topiramate as an initial treatment were predictors of a reduction in oral prophylactics (area under the curve, 70.3%; P < 0.001). CONCLUSIONS: Our study demonstrated the efficacy and safety of onabotulinumtoxinA. This treatment reduced the use of oral prophylactics. Withdrawal of oral prophylactics was most likely to occur after five cycles of treatment.

Psychosocial repercussion of migraine: is it a stigmatized disease?

INTRODUCTION: Stigma manifests both in prejudices and rejection from society towards patients who suffer from a specific pathology, and by patient's internalization of this discrimination, with the consequent repercussions on their state of mind and quality of life. The aim of the study was to quantify the stigma associated with migraine and analyze whether it is related to the clinical-demographic characteristics of the patients, as well as the possible impact on their daily lives. MATERIALS AND METHODS: The stigma scale for chronic illness (SSCI) and other questionnaires were administered to 56 patients with episodic migraine (EM), 18 with chronic migraine (CM), and 21 with epilepsy, as a control group. RESULTS: The mean SSCI score was higher (51.6 ± 15.0) in the CM group than in the EM (45.0 ± 13.5) and epilepsy (47.6 ± 15.5) groups, without reaching statistical significance. In addition, the score was higher in patients who were unemployed, divorced, and in those who had migraine with aura. A statistically significant correlation was found between the SSCI score and the impact of migraine on daily life, the presence of stress, anxiety and depression, and low self-esteem. CONCLUSIONS: There is a stigma around migraine in our society, which seems to be more prevalent in patients with certain socio-demographic characteristics, and that is related to stress, mood alterations, and low self-esteem. Trying to reduce stigma could contribute to improve the control of migraine and reduce the impact of the disease at a socio-economic level.

Prevalence of asthma, allergic rhinitis and pollinosis in a city of Brazil: A monitoring study.

BACKGROUND: Allergic respiratory diseases such as asthma and allergic rhinitis have increased considerably in the last decades. OBJECTIVE: The present study estimates prevalence trends of asthma, allergic rhinitis and pollinosis in the population of a city of Southern Brazil, without restriction of age, from 2011 to 2018, using the ISAAC standardized questionnaire. METHODS: Data was collected from March to June of 2011 and during the same months in 2018, in order to verify trends in the prevalence of these allergic conditions. The total sample consisted of 3132 individuals of both sexes living in the municipality of Santo Ângelo, in the state of Rio Grande do Sul, Brazil. RESULTS: No differences were observed in the prevalence of asthma diagnosis (15.1% in 2011 and 13.8% in 2018), however the prevalence of current wheeze was significantly reduced from 24.7% in 2011 to 21.2% in 2018 (p<0.05). Regarding allergic conditions in 2011 and in 2018, a significant reduction was observed (p<0.001) in reported current rhinitis (63.3% vs. 50.5%), rhinoconjunctivitis (48.9% vs. 38.8%), hay fever (52.0% vs. 43.3%), and pollinosis (29.0% vs 17.0%). Moreover, we observed an inverse relation between age and rhinoconjunctivitis and hay fever, and all symptoms were more frequent in females. Rhinoconjunctivitis and hay fever, as well as current rhinitis and pollinosis were highly prevalent among 30-39 years-old individuals, whereas current wheeze affected mainly the age group 10-19 years-old. CONCLUSION: While the prevalence of asthma remained similar after seven years, allergic rhinitis and pollinosis declined between 2011 and 2018.

Microgravity effects on frozen human sperm samples.

PURPOSE: Microgravity has severe effects on cellular and molecular structures as well as on metabolic interactions. The aim of this study is to investigate the effects of microgravity (µg) exposure on human frozen sperm samples. METHODS: Sibling samples from 15 normozoospermic healthy donors were frozen using glycerol as cryoprotectant and analyzed under microgravity and ground conditions. Microgravity was obtained by parabolic flights using a CAP10B plane. The plane executed 20 parabolic maneuvers with a mean of 8.5 s of microgravity for each parabola. RESULTS: Frozen sperm samples preserved in cryostraws and stored in a secure and specific nitrogen vapor cryoshipper do not suffer significant alterations after µg exposure. Comparing the study group (µg) and the control group (1 g), similar results were obtained in the main parameters studied: sperm motility (M/ml) 13.72 ± 12.57 vs 13.03 ± 12.13 (- 0.69 95% CI [- 2.9; 1.52]), progressive a + b sperm motility (%) 21.83 ± 11.69 vs 22.54 ± 12.83 (0.03 95% CI [- 0.08; 0.15]), sperm vitality (%) 46.42 ± 10.81 vs 44.62 ± 9.34 (- 0.04 95% CI [- 0.13; 0.05]), morphologically normal spermatozoa (%) 7.03 ± 2.61 vs 8.09 ± 3.61 (0.12 95% CI [0.01; 0.24]), DNA sperm fragmentation by SCD (%) 13.33 ± 5.12 vs 13.88 ± 6.14 (0.03 95% CI [- 0.09; 0.16]), and apoptotic spermatozoa by MACS (%) 15.47 ± 15.04 vs 23.80 ± 23.63 (- 0.20 95% CI [- 0.66; 1.05]). CONCLUSION: The lack of differences obtained between frozen samples exposed to µg and those maintained in ground conditions provides the possibility of considering the safe transport of human male gametes to space. Nevertheless, further research is needed to validate the results and to consider the possibility of creating a human sperm bank outside the Earth. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT03760783.

Real world preventative drug management of migraine among Spanish neurologists.

BACKGROUND: Many different preventatives have showed efficacy in the treatment of migraine. National guidelines differ in their recommendations and patients' characteristics are usually taken into account in their selection. In Spain, real life use of preventive therapies seems to be heterogeneous. We aimed to evaluate differences in clinical practice and adherence to national guidelines among Spanish neurologists. METHODS: Observational descriptive study. A survey was conducted among neurologists ascribed to the Spanish Society of Neurology. Participants were differentiated in accordance with their dedication to headache disorders. We analysed socio-demographic parameters and evaluated 43 questions considering migraine management as well as therapeutic choices regarding migraine sub-types and finally, neurologists' personal perception. RESULTS: One hundred fifty-five neurologists participated from 17 different regions, 43.4% of them female and 53.3% under 40 years of age. 34.9% confirmed headache disorders as their main interest. The first choice for preventive therapy in chronic migraine among participants was topiramate (57%) followed by amytriptiline (17.9%) and beta-blockers (14.6%). However in episodic migraine, the preferred options were beta-blockers (47.7%), topiramate (21.5%) and amytriptiline (13.4%). Regarding perceived efficacy, topiramate was considered the best option in chronic migraine (42.7%) followed by onabotulinumtoxinA (25.5%) and amitryptiline (22.4%). Where episodic migraine was concerned, surveyed neurologists perceived topiramate (43.7%) and beta-blockers (30.3%) as the best options. When we evaluated the duration of treatment use with a view to adequate therapeutic response, 43.5% of neurologists preferred 3 months duration and 39.5% were in favour of 6 months duration in episodic migraine. However, considering the preferred duration of treatment use in chronic migraine, 20.4% recommended 3 months, 42.1% preferred 6 months and 12.5% and 22.4% opted for 9 and 12 months respectively. When considering onabotulinumtoxinA therapy, the number of prior therapeutic failures was zero in 7.2% of neurologists, one in 5.9%, two in 44.1%, three in 30.9% and four or more in 11.9%. Following an initial treatment failure with onabotulinumtoxinA, 49% of subjects decided against a second treatment. The number of OnabotA procedures before considering it as ineffective was two in 18.9% of neurologists, three in 70.8% and four in 10.4%. CONCLUSIONS: The initial management of migraine among Spanish Neurologists is in line with most guidelines, where first choice preventative drugs are concerned. The Management of episodic migraine differed from chronic migraine, both in terms of neurologist preference and in their perceived efficacy.

CALCA and TRPV1 genes polymorphisms are related to a good outcome in female chronic migraine patients treated with OnabotulinumtoxinA.

BACKGROUND: Some variables have been proposed as predictors of efficacy of OnabotulinumtoxinA in chronic migraine patients, but data available are inconclusive. We aimed to analyse the influence of single nucleotide polymorphisms in the response to OnabotulinumtoxinA. METHODS: We included 156 female patients treated with OnabotulinumtoxinA accordingly to PREEMPT paradigm in three headache units. OnabotulinumtoxinA was offered to patients that had not responded to topiramate and at least one other preventative. Age at first procedure was 43.7 ± 11.8 years (16-74). Patients with a reduction of at least 50% in the number of migraine days after two OnabotulinumtoxinA procedures were considered as responders. We analysed 25 polymorphisms selected for their relevance regarding migraine pathophysiology and their association with migraine according to previously published genome-wide association studies. Genotyping was performed using KASP probes and a LightCycler-480 (Roche-Diagnostics). Allelic, genotypic frequencies and dominance/recesivity hypothesis of the allelic variants were compared between responders and non-responders by Fisher's exact test. RESULTS: Response to treatment with OnabotulinumtoxinA was achieved in 120 patients (76,9%). Two polymorphisms showed differences: CALCA rs3781719, where allele C represents 26.9% in responders and 40.9% in non-responders (p = 0.007, OR = 3.11 (1.33-7.26)); and TRPV1 rs222749, where allele A represents 4.17% in responders and 12.5% in non-responders (p = 0.013, OR = 3.29 (1.28-8.43)). No significant differences in rest of polymorphisms or clinical or demographic variables were found. CONCLUSIONS: Polymorphic variations of CALCA and TRPV1 genes might play a role as prognostic markers of efficacy of OnabotulinumtoxinA in chronic migraine female patients in our population.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides.

Effective in Vivo Targeting of Influenza Virus through a Cell-Penetrating/Fusion Inhibitor Tandem Peptide Anchored to the Plasma Membrane.

The impact of influenza virus infection is felt each year on a global scale when approximately 5-10% of adults and 20-30% of children globally are infected. While vaccination is the primary strategy for influenza prevention, there are a number of likely scenarios for which vaccination is inadequate, making the development of effective antiviral agents of utmost importance. Anti-influenza treatments with innovative mechanisms of action are critical in the face of emerging viral resistance to the existing drugs. These new antiviral agents are urgently needed to address future epidemic (or pandemic) influenza and are critical for the immune-compromised cohort who cannot be vaccinated. We have previously shown that lipid tagged peptides derived from the C-terminal region of influenza hemagglutinin (HA) were effective influenza fusion inhibitors. In this study, we modified the influenza fusion inhibitors by adding a cell penetrating peptide sequence to promote intracellular targeting. These fusion-inhibiting peptides self-assemble into ∼15-30 nm nanoparticles (NPs), target relevant infectious tissues in vivo, and reduce viral infectivity upon interaction with the cell membrane. Overall, our data show that the CPP and the lipid moiety are both required for efficient biodistribution, fusion inhibition, and efficacy in vivo.

Specificity of electroclinical features in the diagnosis of ring chromosome 20.

BACKGROUND: Ring chromosome 20 (R20) syndrome is a chromosomal disorder characterized mainly by drug-resistant frontal lobe seizures, recurrent nonconvulsive status epilepticus (NCSE), and typical EEG features. The aim of this study was to investigate if this triad is common and specific to all patients with R20. METHODS: In this cross-sectional study (from 2000 to 2011), we selected patients who fulfilled at least two out of three criteria: drug-resistant frontal lobe seizures, recurrent NCSE, and characteristic electroencephalography (EEG) features. In all patients, diagnosis was based on karyotype analysis of at least 100 metaphases. RESULTS: We identified 36 patients who met at least two of the selected criteria: six patients (16.7%) with R20 and 30 (83.3%) without R20 (non-R20). All patients with R20 met all three criteria. Eleven (36.7%) patients without R20, however, also displayed the full triad. In 19 patients without R20 (63.3%), one of the three clinical features was missing: frontal lobe seizures were not resistant to antiepileptic drugs (AED) in four (13.3%), recurrent NCSE was missing in six (20%), and nine (30%) patients did not have typical EEG features. Based on this data, specificity was 63.3%, positive predictive value was 35.3%, and sensitivity and negative predictive values were 100%. Additionally, a review of all publications describing the R20 phenotype revealed that 81.98% of patients with R20 display the full electroclinical triad. CONCLUSIONS: In our study, all patients with R20 displayed the three electroclinical characteristics. This is in line with previous reports (presenting high sensitivity and negative predictive value). However, these features can also be observed in other epilepsies and are not specific to R20. Our findings suggest that in the presence of the full triad of symptoms, karyotype analysis focused on chromosome 20 should be conducted.

Surface damage in cystine, an amino acid dimer, induced by keV ions.

We have studied the interaction of an ion beam (17.6 keV F-) with cystine, a dimer formed by the binding of two cysteine residues. Cystine can be considered as an ideal prototype for the study of the relevance of the disulfide (-S-S-) chemical bond in biomolecules. For the sake of comparison, the amino acid cysteine has also been subjected to the same experimental conditions. Characterization of the samples by XPS and NEXAFS shows that both pristine cystine and pristine cysteine are found as a dipolar ion (zwitterion). Following irradiation, the dimer and the amino acid show a tendency to change from the dipole ion form to the normal uncharged form. The largest spectral modification was observed in the high resolution XPS spectra obtained at around the N 1s core level for the two biomolecules. The 2p sulfur edge spectra of cysteine and cystine were much less sensitive to radiation effects. We suggest that the disulfide bond (-S-S-) remains stable before and after irradiation, contributing to the larger radiation stability of cystine as compared to the amino acid cysteine.

Who are the patients that default tuberculosis treatment? - space matters!

The goals of this article are: (i) to understand how individual characteristics affect the likelihood of patients defaulting their pulmonary tuberculosis (PTB) treatment regimens; (ii) to quantify the predictive capacity of these risk factors; and (iii) to quantify and map spatial variation in the risk of defaulting. We used logistic regression models and generalized additive models with a spatial component to determine the odds of default across continental Portugal. We focused on new PTB cases, diagnosed between 2000 and 2013, and included some individual information (sex, age, residence area, alcohol abuse, intravenous drug use, homelessness, HIV, imprisonment status). We found that the global default rate was 4·88%, higher in individuals with well-known risk profiles (males, immigrants, HIV positive, homeless, prisoners, alcohol and drug users). Of specific epidemiological interest was that our geographical analysis found that Portugal's main urban areas (the two biggest cities) and one tourist region have higher default rates compared to the rest of the country, after adjusting for the previously mentioneded risk factors. The challenge of treatment defaulting, either due to other individual non-measured characteristics, healthcare system failure or patient recalcitrance requires further analysis in the spatio-temporal domain. Our findings suggest the presence of significant within-country variation in the risk of defaulting that cannot be explained by these classical individual risk factors alone. The methods we advocate are simple to implement and could easily be applied to other diseases.

Could monopronucleated ICSI zygotes be considered for transfer? Analysis through time-lapse monitoring and PGS.

PURPOSE: The purpose of this study was to investigate the chromosomal constitution and the developmental potential of intracytoplasmic sperm injection (ICSI) deriving embryos displaying a single pronucleus at the zygote stage. METHODS: Eighty-eight embryos from single pronucleus (1PN) two polar bodies (2PB) ICSI zygotes from 64 preimplantational genetic screening (PGS) cycles (October 2012-December 2014), were retrospectively analyzed. Zygotes were cultured in a time-lapse incubator. Embryo biopsy was performed on day 3 and genetic analysis approached by array comparative genomic hybridization. RESULTS: Chromosomal analysis revealed that 17% (15/88) of embryos derived from 1PN 2PB zygotes were diagnosed as euploid. After blastomere biopsy at day 3, the blastocyst rate at day 5 was 3.4% (3/88). Only 2.3% (2/88) euploid blastocysts were obtained. In two couples and after counseling and patient agreement, the transfer of a euploid blastocyst from a 1PN 2PB ICSI zygote was performed resulting in the birth of a healthy child. CONCLUSIONS: These results open the possibility to consider embryos coming from 1PN 2PB ICSI zygotes for transfer when no other embryos from 2PN 2PB ICSI zygotes are available and if a PGS diagnosis of euploidy is obtained. Confirmation of biparental inheritance is strongly recommended.

Molecular markers of putative spermatogonial stem cells in the domestic cat.

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43 days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

STUDY HYPOTHESIS: Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? STUDY FINDING: We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. WHAT IS KNOWN ALREADY: Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. LIMITATIONS, REASONS FOR CAUTION: All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest.

Stem cells in reproductive medicine: ready for the patient?

STUDY QUESTION: Are there effective and clinically validated stem cell-based therapies for reproductive diseases? SUMMARY ANSWER: At the moment, clinically validated stem cell treatments for reproductive diseases and alterations are not available. WHAT IS KNOWN ALREADY: Research in stem cells and regenerative medicine is growing in scope, and its translation to the clinic is heralded by the recent initiation of controlled clinical trials with pluripotent derived cells. Unfortunately, stem cell 'treatments' are currently offered to patients outside of the controlled framework of scientifically sound research and regulated clinical trials. Both physicians and patients in reproductive medicine are often unsure about stem cells therapeutic options. STUDY DESIGN, SIZE, DURATION: An international working group was assembled to review critically the available scientific literature in both the human species and animal models. PARTICIPANTS/MATERIALS, SETTING, METHODS: This review includes work published in English until December 2014, and available through Pubmed. MAIN RESULTS AND THE ROLE OF CHANCE: A few areas of research in stem cell and reproductive medicine were identified: in vitro gamete production, endometrial regeneration, erectile dysfunction amelioration, vaginal reconstruction. The stem cells studied range from pluripotent (embryonic stem cells and induced pluripotent stem cells) to monopotent stem cells, such as spermatogonial stem cells or mesenchymal stem cells. The vast majority of studies have been carried out in animal models, with data that are preliminary at best. LIMITATIONS, REASONS FOR CAUTION: This review was not conducted in a systematic fashion, and reports in publications not indexed in Pubmed were not analyzed. WIDER IMPLICATIONS OF THE FINDINGS: A much broader clinical knowledge will have to be acquired before translation to the clinic of stem cell therapies in reproductive medicine; patients and physicians should be wary of unfounded claims of improvement of existing medical conditions; at the moment, effective stem cell treatment for reproductive diseases and alterations is not available. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: NA.

Expression of genes related to tolerance to low temperature for maize seed germination.

The aim of this study was to characterize maize lines tolerant to cold temperatures during the germination process. Seeds from lines with different levels of tolerance to low temperatures were used; 3 lines were classified as tolerant and 3 as susceptible to low germination temperatures. A field was set up to multiply seeds from selected lines. After the seeds were harvested and classified, we conducted physiological tests and analyzed fatty acid content of palmitic, stearic, oleic, linoleic, linolenic, and eicosenoic acids. In proteomic analysis, the expression of heat-resistant proteins, including catalase, peroxidase, esterase, superoxide dismutase, and α-amylase, were evaluated. Transcript analysis was used to measure the expression of the genes AOX1, AOX2, ZmMPK-17, and ZmAN-13. The material showing the highest susceptibility to low germination temperatures contained high saturated fatty acid content. Expression of α-amylase in seeds soaked for 72 h at a temperature of 10°C was lower than expression of α-amylase when soaked at 25°C for the same amount of time. We observed variation in the expression of heat-resistant proteins in seeds of the lines evaluated. The genes AOX and Zm-AN13 were promising for use in identifying maize materials that are tolerant to low germination temperatures.

Verification and characterization of chromosome duplication in haploid maize.

Doubled haploid technology has been used by various private companies. However, information regarding chromosome duplication methodologies, particularly those concerning techniques used to identify duplication in cells, is limited. Thus, we analyzed and characterized artificially doubled haploids using microsatellites molecular markers, pollen viability, and flow cytometry techniques. Evaluated material was obtained using two different chromosome duplication protocols in maize seeds considered haploids, resulting from the cross between the haploid inducer line KEMS and 4 hybrids (GNS 3225, GNS 3032, GNS 3264, and DKB 393). Fourteen days after duplication, plant samples were collected and assessed by flow cytometry. Further, the plants were transplanted to a field, and samples were collected for DNA analyses using microsatellite markers. The tassels were collected during anthesis for pollen viability analyses. Haploid, diploid, and mixoploid individuals were detected using flow cytometry, demonstrating that this technique was efficient for identifying doubled haploids. The microsatellites markers were also efficient for confirming the ploidies preselected by flow cytometry and for identifying homozygous individuals. Pollen viability showed a significant difference between the evaluated ploidies when the Alexander and propionic-carmin stains were used. The viability rates between the plodies analyzed show potential for fertilization.

Decoding distinct membrane interactions of HIV-1 fusion inhibitors using a combined atomic force and fluorescence microscopy approach.

Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15µM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides.