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Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (Vm), and modulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient Vm hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology.

The cause of structural valve deterioration (SVD) is unclear. Therefore, we investigated oxidative stress markers in sera from patients with bioprosthetic heart valves (BHVs) and their association with SVD. Blood samples were taken from SVD (Phase A) and BHV patients during the first 24 (Phase B1) and >48 months (Phase B2) after BHV implantation to assess total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrotyrosine (NT). The results show that MDA levels increased significantly 1 month after surgery in all groups but were higher at 6 months only in incipient SVD patients. NT levels increased gradually for the first 24 months after implantation in the BHV group. Patients with transcatheter aortic valve implantation (TAVI) showed even higher levels of stress markers. After >48 months, MDA and NT continued to increase in BHV patients with a further elevation after 60-72 months; however, these levels were significantly lower in the incipient and established SVD groups. In conclusion, oxidative stress may play a significant role in SVD, increasing early after BHV implantation, especially in TAVI cases, and also after 48 months' follow-up, but decreasing when SVD develops. Oxidative stress potentially represents a target of therapeutic intervention and a biomarker of BHV dysfunction.

This music score was submitted for the Kaleidoscope 2020 Call for Scores, an open access collaboration with the UCLA Music Library.

This music score was submitted for the Kaleidoscope 2020 Call for Scores, an open access collaboration with the UCLA Music Library.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study-ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)-providing a template for the assessment of ART procedures in projects involving other endangered species.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Il saggio ripercorre la storia del significato del termine “Lombardia”, dalla sua formazione nell’alto medioevo alle differenti valenze del concetto storico-politico e geografico; usato inizialmente dai bizantini per indicare le popolazioni italiche sottratte al loro controllo dai longobardi, il termine si andò lentamente cristallizzando in riferimento agli abitanti della Langobardia maior che, dal tardo XII secolo, presero ad essere chiamati “lombardi”, anche in chiave anti imperiale, con un’accezione geografica per distinguerli dai longobardi delle Langobardia minor dei ducati di Spoleto e Benevento., The essay traces the history of the meaning of the term "Lombardy", from its formation in the early Middle Ages to the different values of the historical-political and geographical concept; initially used by the Byzantines to indicate the Italic populations removed from their control by the Lombards, the term slowly crystallized in reference to the inhabitants of Langobardia maior who, from the late 12th century, began to be called "Lombard", with a geographical meaning to distinguish them from the Lombards of the Langobardia minor of the duchies of Spoleto and Benevento.

Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project's partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

Introduction: Previous studies showed that quarantine for pandemic diseases is associated with several psychological and medical effects. The consequences of quarantine for COVID-19 pandemic in patients with dementia are unknown. We investigated the clinical changes in patients with Alzheimer’s disease and other dementias, and evaluated caregivers’ distress during COVID-19 quarantine. Methods: The study involved 87 Italian Dementia Centers. Patients with Alzheimer’s Disease (AD), Dementia with Lewy Bodies (DLB), Frontotemporal Dementia (FTD), and Vascular Dementia (VD) were eligible for the study. Family caregivers of patients with dementia were interviewed by phone in April 2020, 45 days after quarantine declaration. Main outcomes were patients’ changes in cognitive, behavioral, and motor symptoms. Secondary outcomes were effects on caregivers’ psychological features. Results: 4913 patients (2934 females, 1979 males) fulfilled the inclusion criteria. Caregivers reported a worsening in cognitive functions in 55.1% of patients, mainly in subjects with DLB and AD. Aggravation of behavioral symptoms was observed in 51.9% of patients. In logistic regression analysis, previous physical independence was associated with both cognitive and behavioral worsening (odds ratio 1.85 [95% CI 1.42–2.39], 1.84 [95% CI 1.43–2.38], respectively). On the contrary, pandemic awareness was a protective factor for the worsening of cognitive and behavioral symptoms (odds ratio 0.74 [95% CI 0.65–0.85]; and 0.72 [95% CI 0.63–0.82], respectively). Approximately 25.9% of patients showed the onset of new behavioral symptoms. A worsening in motor function was reported by 36.7% of patients. Finally, caregivers reported a high increase in anxiety, depression, and distress. Conclusion: Our study shows that quarantine for COVID-19 is associated with an acute worsening of clinical symptoms in patients with dementia as well as increase of caregivers’ burden. Our findings emphasize the importance to imp

Background: Family caregivers of patients with dementia are at high risk of stress and burden, and quarantine due to the coronavirus disease 2019 (COVID-19) pandemic may have increased the risk of psychological disturbances in this population. The current study was carried out during the national lockdown declared in March 2020 by the Italian government as a containment measure of the first wave of the coronavirus pandemic and is the first nationwide survey on the impact of COVID-19 lockdown on the mental health of dementia informal caregivers. Methods: Eighty-seven dementia centers evenly distributed on the Italian territory enrolled 4,710 caregiver-patient pairs. Caregivers underwent a telephone interview assessing classical symptoms of caregiver stress and concern for the consequences of COVID-19 infection on patient's health. We calculated prevalence of symptoms and regressed them on various potential stress risk factors: caregivers' sociodemographic characteristics and lifestyle, patients' clinical features, and lockdown-related elements, like discontinuity in medical care. Results: Approximately 90% of caregivers reported at least one symptom of stress, and nearly 30% reported four or more symptoms. The most prevalent symptoms were concern for consequences of COVID-19 on patient's health (75%) and anxiety (46%). The main risk factors for stress were identified as a conflicting relationship with the patient and discontinuity in assistance, but caregiver's female sex, younger age, lower education, and cohabitation with the patient also had an impact. Availability of help from institutions or private individuals showed a protective effect against sense of abandonment but a detrimental effect on concern about the risk for the patient to contract COVID-19. The only protective factor was mild dementia severity, which was associated with a lower risk of feeling isolated and abandoned; type of dementia, on the other hand, did not affect stress risk. Conclusion: Our re

Il vaccino antinfluenzale Fluenz Tetra® (vaccino vivo attenuato quadrivalente, spray nasale, in sospensione) sarà disponibile in Italia per la prevenzione e il controllo dell’influenza per la stagione influenzale 2021-2022. Tale vaccino è indicato per la profilassi dell’influenza in bambini e adolescenti di età compresa tra i 24 mesi e i 18 anni [1]. I decision makers dovranno, quindi, presto valutarne l’utilizzo. Questa decisione potrebbe essere supportata dalla disponibilità di uno specifico rapporto di Health Technology Assessment (HTA). Infatti, l’HTA rappresenta oggi l’approccio valutativo più utile per i decision makers per prendere le più appropriate decisioni in ambito sanitario in base alle prove scientifiche disponibili. Anche il Piano Nazionale Prevenzione Vaccinale 2017-2019 (PNPV 2017-2019) conferma il ruolo rilevante dell’HTA ai fini della valutazione delle potenzialità e dei limiti di un vaccino e auspica un sempre maggiore utilizzo di questa metodologia per favorire le decisioni basate sulle evidenze, in un’ottica di corretto utilizzo delle limitate risorse disponibili [2]. In ambito vaccinale l’HTA si esplica nella valutazione dell’epidemiologia della specifica infezione e delle malattie correlate nella popolazione target, del disease burden della patologia, delle attuali misure preventive e terapeutiche disponibili contro l'infezione e le malattie esaminate, dell’efficacia e sicurezza del vaccino oggetto di studio e degli eventuali altri vaccini disponibili, delle valutazioni economiche, e infine degli aspetti etici, legali, sociali e organizzativi correlati all’introduzione di tale vaccino [3]. Pertanto, tutte le evidenze scientifiche disponibili su questi aspetti dovrebbero essere raccolte e esaminate criticamente all’interno di un rapporto di HTA al fine di eseguire una valutazione completa. Particolare attenzione va posta sul fatto che questa metodologia di valutazione si applica non solo per la valutazione dell’introduzione di nuovi vac

Background: Family caregivers of patients with dementia are at high risk of stress and burden, and quarantine due to the coronavirus disease 2019 (COVID-19) pandemic may have increased the risk of psychological disturbances in this population. The current study was carried out during the national lockdown declared in March 2020 by the Italian government as a containment measure of the first wave of the coronavirus pandemic and is the first nationwide survey on the impact of COVID-19 lockdown on the mental health of dementia informal caregivers. Methods: Eighty-seven dementia centers evenly distributed on the Italian territory enrolled 4,710 caregiver-patient pairs. Caregivers underwent a telephone interview assessing classical symptoms of caregiver stress and concern for the consequences of COVID-19 infection on patient's health. We calculated prevalence of symptoms and regressed them on various potential stress risk factors: caregivers' sociodemographic characteristics and lifestyle, patients' clinical features, and lockdown-related elements, like discontinuity in medical care. Results: Approximately 90% of caregivers reported at least one symptom of stress, and nearly 30% reported four or more symptoms. The most prevalent symptoms were concern for consequences of COVID-19 on patient's health (75%) and anxiety (46%). The main risk factors for stress were identified as a conflicting relationship with the patient and discontinuity in assistance, but caregiver's female sex, younger age, lower education, and cohabitation with the patient also had an impact. Availability of help from institutions or private individuals showed a protective effect against sense of abandonment but a detrimental effect on concern about the risk for the patient to contract COVID-19. The only protective factor was mild dementia severity, which was associated with a lower risk of feeling isolated and abandoned; type of dementia, on the other hand, did not affect stress risk. Conclusion: Our re

Introduction: Previous studies showed that quarantine for pandemic diseases is associated with several psychological and medical effects. The consequences of quarantine for COVID-19 pandemic in patients with dementia are unknown. We investigated the clinical changes in patients with Alzheimer’s disease and other dementias, and evaluated caregivers’ distress during COVID-19 quarantine. Methods: The study involved 87 Italian Dementia Centers. Patients with Alzheimer’s Disease (AD), Dementia with Lewy Bodies (DLB), Frontotemporal Dementia (FTD), and Vascular Dementia (VD) were eligible for the study. Family caregivers of patients with dementia were interviewed by phone in April 2020, 45 days after quarantine declaration. Main outcomes were patients’ changes in cognitive, behavioral, and motor symptoms. Secondary outcomes were effects on caregivers’ psychological features. Results: 4913 patients (2934 females, 1979 males) fulfilled the inclusion criteria. Caregivers reported a worsening in cognitive functions in 55.1% of patients, mainly in subjects with DLB and AD. Aggravation of behavioral symptoms was observed in 51.9% of patients. In logistic regression analysis, previous physical independence was associated with both cognitive and behavioral worsening (odds ratio 1.85 [95% CI 1.42–2.39], 1.84 [95% CI 1.43–2.38], respectively). On the contrary, pandemic awareness was a protective factor for the worsening of cognitive and behavioral symptoms (odds ratio 0.74 [95% CI 0.65–0.85]; and 0.72 [95% CI 0.63–0.82], respectively). Approximately 25.9% of patients showed the onset of new behavioral symptoms. A worsening in motor function was reported by 36.7% of patients. Finally, caregivers reported a high increase in anxiety, depression, and distress. Conclusion: Our study shows that quarantine for COVID-19 is associated with an acute worsening of clinical symptoms in patients with dementia as well as increase of caregivers’ burden. Our findings emphasize the importance to imp

Background: In March 2020, the World Health Organization declared a global pandemic due to the novel coronavirus SARS-CoV-2 and several governments planned a national quarantine in order to control the virus spread. Acute psychological effects of quarantine in frail elderly subjects with special needs, such as patients with dementia, have been poorly investigated. The aim of this study was to assess modifications of neuropsychiatric symptoms during quarantine in patients with dementia and their caregivers. Methods: This is a sub-study of a multicenter nation-wide survey. A structured telephone interview was delivered to family caregivers of patients with diagnosis of Alzheimer disease (AD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), and vascular dementia (VD), followed regularly at 87 Italian memory clinics. Variations in behavioral and psychological symptoms (BPSD) were collected after 1 month since quarantine declaration and associations with disease type, severity, gender, and caregiver’s stress burden were analyzed. Results: A total of 4,913 caregivers participated in the survey. Increased BPSD was reported in 59.6% of patients as worsening of preexisting symptoms (51.9%) or as new onset (26%), and requested drug modifications in 27.6% of these cases. Irritability, apathy, agitation, and anxiety were the most frequently reported worsening symptoms and sleep disorder and irritability the most frequent new symptoms. Profile of BPSD varied according to dementia type, disease severity, and patients’ gender. Anxiety and depression were associated with a diagnosis of AD (OR 1.35, CI: 1.12–1.62), mild to moderate disease severity and female gender. DLB was significantly associated with a higher risk of worsening hallucinations (OR 5.29, CI 3.66–7.64) and sleep disorder (OR 1.69, CI 1.25–2.29), FTD with wandering (OR 1.62, CI 1.12–2.35), and change of appetite (OR 1.52, CI 1.03–2.25). Stress-related symptoms were experienced by two-thirds of caregiv

Background & aims: Clinical pig islet transplantation for the treatment of type I diabetes is still hindered by obvious immunological considerations and less investigated physiological incompatibilities. Cellular encapsulation, donor genetic engineering and host immunomodulation can help improve islet survival in a xenotransplantation context but adequate insulin output from transplanted islets remains the ultimate goal to be achieved for islet transplantation to be clinically efficient. We have previously shown that adenovirus-driven expression of a cassette carrying a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in isolated neonatal and adult pig islets significantly increases their secretory response to in vitro glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to replicate our previous results in a transgenic pig model with beta-cell specific expression of our GLP1M3R cassette, to characterize in vivo and in vitro islet function of these pigs and to verify whether their offspring exhibit the same improved insulin secretion as founder animals. Material & methods: Cloned transgenic pigs were produced using Talens technology and validated by TLA sequencing analyses. Pancreata were collected from 14-day old piglets. Insulin secretion in response to different stimuli was evaluated during dynamic islet perifusion experiments. After reaching adult age, selected animals were subjected to IVGTT to study in vivo islet function. Transgenic animals were then bred and islet function of their offspring was studied as for founder animals. Results: Transgenic cloned pig founders (male and female) expressing the GLP1M3R cassette at the beta cell level were successfully obtained. Islets isolated from these piglets showed a 5.5 to 7.5-fold increase of their insulin output upon stimulation with 15 mM glucose. They maintained regul

The main benefit of xenotransplantation is its potential to overcome the worldwide organ shortage experienced in allotransplantation. Allogeneic transplantation is the only successful therapy for several life-threatening diseases, with cell, tissue or organ donation only partially meeting the demand and many patients dying while waiting for treatment. With supply falling short of demand, it is foreseen that the use of porcine material may at some stage overcome the existing gap between organ availability and clinical need. Recently, pig islet cells have been utilised in clinical trials, with safety being demonstrated. Indeed, pig-derived cells present several advantages: i) porcine cells have a stable function and differentiation pattern and are not tumorigenic; ii) pig cells have been shown to meet the physiological needs in large animal models; iii) the source of pig cells can be scaled up to meet demands in a highly standardised manner, and with respect to animal welfare regulations; iv) ‘designated-pathogen-free’ (DPF) pig lines can be produced, which could result in a higher safety profile than allotransplantation itself; v) the risk of zoonosis, which was raised years ago as the major hurdle, has been recently circumvented and is actually viewed as a controlled risk; and vi) immune risks are being circumvented via the use of genetically modified donor animals and encapsulation of porcine cells, particularly for the treatment of diabetes. Overall, the benefit appears to outweigh potential risks with respect to cellular xenotransplantation and this is discussed further in this review.

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species.

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency’s recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency’s proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

BACKGROUND/OBJECTIVES: Blood polyunsaturated fatty acids (PUFA) are involved in allergy development, but the etiological role of n-6 and n-3 PUFA is still controversial. A European multicenter study of children (IDEFICS) provided the opportunity to explore the cross-sectional association between fatty acids (FA) and allergy. SUBJECTS/METHODS: Blood FA levels were measured in 2600 children aged 2-9 years and were recorded as the percentage of weight of all FA detected. Logistic regression of allergy status on FA components was adjusted for age, sex, country, body mass index, family history of allergic disease, breast-feeding, and number of siblings. The results were given as odds ratios (OR) for current vs no allergy ever and an increase in FA by 1 s.d. RESULTS: Overall, higher proportions of n-6 PUFA were associated with higher odds of allergy (OR = 1.21 (1.05, 1.40)). Monounsaturated FA (MUFA) were associated with reduced risk for allergy (OR = 0.75 (0.65, 0.87)), whereas saturated FA did not differ by allergy status. The strongest associations were observed in children < 4 years old, with ORs of allergy given as 1.62 (1.15, 2.29) for n-3 PUFA and 0.63 (0.42, 0.95) for MUFA. With regard to individual FA, these associations were independently observed for docosapentaenoic acid (22: 5 n-3) and oleic acid (18: 1 n-9). CONCLUSIONS: Both PUFA subtypes were positively associated with allergy in an age-dependent manner, whereas MUFA was associated with less allergy. The observation of high proportions of n-3 PUFA in allergic children younger than 4 years might help to understand the nature of early onset of atopic disease., Group Author(s): IDEFICS Consortium© 2017 Macmillan Publishers Limited, part of Springer Nature

BACKGROUND/OBJECTIVES: Fatty acids are hypothesized to influence cardiovascular disease risk because of their effect on inflammation. The aim of this study is to assess the relationship between whole-blood fatty acids (WBFAs) and high-sensitivity C-reactive protein (hs-CRP) in European children. SUBJECTS/METHODS: A total of 1401 subjects (697 boys and 704 girls) aged between 2 and 9 years from the IDEFICS (Identification and prevention of Dietary- and lifestyle-induced health EFfects in Children and infantS) study were measured in this cross-sectional analysis. The sample was divided into three categories of hs-CRP. Associations between WBFA and hs-CRP were assessed by logistic regression models adjusting for body mass index (BMI), country, age, breastfeeding, mother's education and hours of physical activity. RESULTS: Linoleic acid (LA) (P = 0.013, 95% confidence interval (CI):0.822-0.977) and sum of n-6 WBFA (P = 0.029, 95% CI: 0.866-0.992) concentrations were associated with lower concentrations of hs-CRP in boys. In girls, a high ratio of eicosapentaenoic acid (EPA)/arachidonic acid (AA) was associated (P = 0.018, 95% CI: 0.892-0.989) with lower hs-CRP concentrations. In contrast, sum of blood n-6 highly unsaturated fatty acids (P = 0.012, 95% CI: 1.031-1.284), AA (P = 0.007, 95% CI: 1.053-1.395) and AA/LA ratio (P = 0.005, 95% CI: 1.102-1.703) were associated (P < 0.05) with higher concentrations of hs-CRP in girls. CONCLUSIONS: The n-6 WBFAs (sum of n-6 FA and LA) were associated with lower hs-CRP in boys and with higher hs-CRP in girls (AA, sum of n-6 highly unsaturated and AA/LA ratio). More studies are needed to identify the optimal levels of WBFAs to avoid low-grade inflammation in children considering the differences by sex and BMI., Group Author(s): IDEFICS Consortium

Il contributo presenta una lettura commentata di Purgatorio II.

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a ligninrelated phenol of industrial interest. In total, more than 15 000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets.

The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not

OBJECTIVES: To establish reference values for fatty acids (FA) especially for n-3 and n-6 long-chain polyunsaturated FAs (LC PUFA) in whole-blood samples from apparently healthy 3-8-year-old European children. The whole-blood FA composition was analysed and the age-and sex-specific distribution of FA was determined. DESIGN AND SUBJECTS: Blood samples for FA analysis were taken from 2661 children of the IDEFICS (identification and prevention of dietary-and lifestyle-induced health effects in children and infants) study cohort. Children with obesity (n = 454) and other diseases that are known to alter the FA composition (n = 450) were excluded leaving 1653 participants in the reference population. MEASUREMENTS: The FA composition of whole blood was analysed from blood drops by a rapid, validated gas chromatographic method. RESULTS: Pearson correlation coefficients showed an age-dependent increase of C18:2n-6 and a decrease of C18:1n-9 in a subsample of normal weight boys and girls. Other significant correlations with age were weak and only seen either in boys or in girls, whereas most of the FA did not show any age dependence. For age-dependent n-3 and n-6 PUFA as well as for other FA that are correlated with age (16:0, C18:0 and C18:1n-9) percentiles analysed with the general additive model for location scale and shape are presented. A higher median in boys than in girls was observed for C20:3n-6, C20:4n-6 and C22:4n-6. CONCLUSIONS: Given the reported associations between FA status and health-related outcome, the provision of FA reference ranges may be useful for the interpretation of the FA status of children in epidemiological and clinical studies., Group Author(s): IDEFICS Consortium

Recently site specific nucleases (ZFN, Tal Effector and CRISPR) emerged as powerful tools for gene modification of different cells types and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010; Whyte et al. 2010). Previously (Brunetti et al. 2008, Clon. Stem. Cells) we generated an EGFP transgenic porcine line (Verro2GFP) characterized by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, mendelian transgene transmission and expression in F1. The aim of this work was to modify a transcriptionally active GFP-locus into one suitable for Cre-mediated RMCE, using EGFP-specific ZFNs. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) DsRedT4 reporter gene between LHA and RHA sequences, we generated the targeting/RMCE vector (pB50 30 DsRed4-PL) and its positive control (C?) for PCR set-up (100–1,000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM ? M199(1:1) ? 10 %FCS, bFGF in 5 %CO2, 5 %O2, were transfected using Nucleofector (V-024 program). In ZFNs-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr) and 2 lg of pB50 30 RedT4-PL/NotI vector were used to 00nucleofect00 1x106 Verro2GFP fibroblasts. Part of transfected cells were plated (3,000 cells/plate) in 10 Petri dishes ([ = 100 mm) and cultured without selection till well growing colonies appeared. Four RFP?/GFPcolonies were identified using epifluorescence, re-plated in two 150 mm dishes/colony and PCR verified. Finally 3 colonies were used in zona-free SCNT to produce reconstructed embryos, and 159 D6 blastocysts/compacted morulae were transferred into two synchronized sows that both became pregnant. One pregnancy is ongoing, another was interrupted at D58 and 7 fetuses (61.14 g ± 17.98 g) were used to generate RFP primary cell lines, finally verified b

Site-specific nucleases (ZFN, Tal effector nucleases and CRISPRs) boosted the genome editing of different species, and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). Previously (Brunetti et al., 2008) we generated an EGFP transgenic porcine line (V2G) characterized by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission and expression in F1. The aim of this work was to generate live cloned piglets using modified cells after two site-specific modifications (ZFNs and RMCE) on the transcriptionally active GFP-locus (V2G), avoiding any cell rejuvenation by SCNT. Homology arms for promoterless targeting vector were derived from pCAGGSEGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) puromycin and hygromycin resistance coding sequences between LHA and RHA sequences, and we generated the targeting/RMCE vectors (pB50 30 Puro-PL and pB50 30 Hygro-PL) and theirs positive controls (C+) for PCR set-up (100–1,000 plasmid copies). V2G fibroblasts cultured in DMEM + M199(1:1) +10 %FCS, bFGF in 5 %CO2, 5 %O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr ) and 2 lg of pB50 30 Puro-PL/KpnI vector, were used to ‘nucleofect’ 0.9 9 106 Verro2GFP fibroblasts. Transfected cells were cultured under puromycin selection (2 lg/ml) for 8 days, and 0.45 9 106 cells were used for the Cre-mediated cassette exchange (1 lg pB50 30 Hygro-PL/KpnI + 2 lg pCAG:CreEGFP). Transfected cells were plated in 10 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 lg/ml) for 12 days, picked up and expanded in 24 multi-well plates for SCNT. Thirty-one colonies were PCR screened for hygromycin and 16 (51.6 %) colonies were positive for Cre-mediated targeted insertion. Four colonies were use

HPV vaccination is expected to reduce the incidence of cervical cancer. The greatest and the earliest health gains will be ensured by high vaccine coverage among all susceptible people. The high costs and the risk of a reduced cost/effectiveness ratio in sexually active girls still represent the main obstacles for a more widespread use of HPV vaccination in many countries. Data on the rate, risk factors, and HPV types in sexually active women could provide information for the evaluation of vaccination policies extended to broader age cohorts. Sexually active women aged 13-26 years enrolled in an Italian cohort study were screened for cervical HPV infections; HPV-DNA positive samples were genotyped by InnoLipa HPV Genotyping Extra or by RFLP genotype analysis. Among the 796 women meeting the inclusion criteria, 10.80% (95% CI 8.65-12.96) were HPV-DNA infected. Age >18 years, lifetime sexual partners >1, and history of STIs were associated to higher risk of HPV infection in the multivariable models adjusted for age, lifetime sexual partners, and time of sexual exposure. The global prevalence of the four HPV vaccine-types was 3.02% (95% CI 1.83-4.20) and the cumulative probability of infection from at least one vaccine-type was 12.82% in 26-years-old women and 0.78% in 18-years-old women. Our data confirm most of the previously reported findings on the risk factors for HPV infections. The low prevalence of the HPV vaccine-types found may be useful for the evaluation of the cost/efficacy and the cost/effectiveness of broader immunization programs beyond the 12-years-old cohort. © 2014 Landes Bioscience.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of trea