Controlling the reactivity of phthalonitriles for the efficient synthesis of chiral phthalocyanines with self-assembly abilities

Binaphthol-bridged phthalonitriles are common precursors for the preparation of chiral phthalocyanines (Pcs). However, self-condensation of these phthalonitriles usually leads to the target Pcs in extremely low yields. In this work, we report a smart approach to enhance the reactivity towards self-condensation of binaphthol-bridged phthalonitriles, which relies on the addition of another highly reactive phthalonitrile that initiates the macrocyclization process, but it is eventually released. After optimization of the reaction conditions, ethynyl-containing binaphthyloxy-bridged Zn(II)Pc (AA)2-2 has been obtained in remarkable yields. This Pc synthon has been further endowed with hydrophilic cationic tails, giving the strongly amphiphilic Zn(II)Pc 1, which has shown the ability to form nanostructures in aqueous solutionsFinancial support from the Spanish MCIN/AEI/10.13039/ 501100011033 (PID2020-116490GB-I00, PID2020- 115801RB-C21), the Comunidad de Madrid and the Spanish State through the Recovery, Transformation and Resilience Plan [“Materiales Disruptivos Bidimensionales (2D)” (MAD2D-CM) (UAM1)-MRR Materiales Avanzados], and the European Union through the Next Generation EU funds is acknowledged. IMDEA Nanociencia acknowledges support from the “Severo Ochoa” Programme for Centres of Excellence in R&D (MINECO, Grant SEV2016-0686

Human keratinocytes are vanilloid resistant

BACKGROUND: Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca(2+)-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+)-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+)-cytotoxicity ([RTX]>15 microM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+)-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials

Effect of follicle size on hormonal status of follicular fluid, oocyte ATP content, and in vitro embryo production in prepubertal sheep

The aim of this study was to assess the effect of follicular size on estradiol (E2) and progesterone (P4) levels in intrafollicular fluid, ATP content in oocytes, and the embryo development rate in prepubertal sheep. Slaughterhouse ovaries were dissected to recover the follicles, which were classified according to the follicle diameter as <3 mm (n = 20) and ≥3 mm (n = 17). Then, follicular fluid was obtained and analyzed by radioimmunoassay to determine the E2 and P4 concentrations. Another group of ovaries was used to recover cumulus-oocyte complexes according to follicle size. In vitro maturation (IVM), in vitro fertilization (IVF), and embryo culture were performed using standard procedures, and ATP level was assessed at 0 and 24 h of IVM. Intrafollicular concentrations of E2 and P4 and E2:P4 ratio were higher in ≥3 mm (18.7 ± 5.9 ng/mL, 7.8 ± 1.2 ng/mL, and 3.6 ± 1.3, respectively) than <3 mm (1.8 ± 0.4 ng/mL, 2.6 ± 0.3 ng/mL and 0.9 ± 0.3, respectively) follicles. The rate of ATP increased during IVM and was higher in oocytes from ≥3 mm than <3 mm (22.4 ± 0.7 and 8.6 ± 2.2-fold change; respectively) follicles. After IVF, the blastocyst development was higher in oocytes recovered from ≥3 mm (11.1 ± 0.9%) than from <3 mm (6.5 ± 0.7%) follicles. These results indicate an improvement in the competence and development of oocytes from ≥3 mm follicles with a higher E2:P4 ratio. Thus, this ratio could be used as reference to design IVM medium and to enhance the in vitro embryo production in lambs

Self-consistent field theory for the interactions between keratin intermediate filaments

Background: Keratins are important structural proteins found in skin, hair and nails. Keratin Intermediate Filaments are major components of corneocytes, nonviable horny cells of the Stratum Corneum, the outermost layer of skin. It is considered that interactions between unstructured domains of Keratin Intermediate Filaments are the key factor in maintaining the elasticity of the skin. Results: We have developed a model for the interactions between keratin intermediate filaments based on self-consistent field theory. The intermediate filaments are represented by charged surfaces, and the disordered terminal domains of the keratins are represented by charged heteropolymers grafted to these surfaces. We estimate the system is close to a charge compensation point where the heteropolymer grafting density is matched to the surface charge density. Using a protein model with amino acid resolution for the terminal domains, we find that the terminal chains can mediate a weak attraction between the keratin surfaces. The origin of the attraction is a combination of bridging and electrostatics. The attraction disappears when the system moves away from the charge compensation point, or when excess small ions and/or NMF-representing free amino acids are added. Conclusions: These results are in concordance with experimental observations, and support the idea that the interaction between keratin filaments, and ultimately in part the elastic properties of the keratin-containing tissue, is controlled by a combination of the physico-chemical properties of the disordered terminal domains and the composition of the medium in the inter-filament region. Keywords: Stratum corneum, Skin keratins, Intermediate filaments, Unstructured terminal domains, Bridging attractio

2013 Report on the State of the Art of Rare Disease Activities in Europe (Ireland)

This document has been produced by the Scientific Secretariat of the European Union Committee ofExperts on Rare Diseases (EUCERD, formerly the European Commission’s Rare Diseases Task Force)through the EUCERD Joint Action: Working for Rare Diseases (N° 2011 22 01, Coordinator: KateBushby, University of Newcastle, United Kingdom), within the European Union’s Second Programmeof Community Action in the Field of Health. More information on the European Union Committee of Experts on Rare Diseases can be found atwww.eucerd.eu

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

In vitro embryo production success in juvenile animals is compromised due to their intrinsic lower oocyte quality. Conventional in vitro maturation (IVM) impairs oocyte competence by inducing spontaneous meiotic resumption. A series of experiments were performed to determine if maintaining meiotic arrest during a pre-maturation culture phase (pre-IVM) prior to conventional IVM improves oocyte competence of juvenile-goat (2 months old) cumulus-oocyte complexes (COCs). In experiment 1, COCs were cultured with C-type natriuretic peptide (CNP; 0, 50, 100, 200 nM) for 6 and 8 h. Nuclear stage was assessed, revealing no differences in the incidence of germinal vesicle (GV) breakdown. In experiment 2, the same CNP concentrations were assessed plus 10 nM estradiol, the known upstream agonist activating expression of NPR2, the exclusive receptor of CNP. CNP (200 nM) plus estradiol increased the rate of oocytes at GV stage at 6 h compared to control group (74.7% vs 28.3%; P<0.05) with predominantly condensed chromatin configuration. In experiment 3, relative mRNA quantification revealed NPR2 expression was down-regulated after pre-IVM (6 h). In experiment 4, analysis of transzonal projections indicated that pre-IVM maintained cumulus-oocyte communication after oocyte recovery. For experiments 5 and 6, biphasic IVM (6 h pre-IVM with CNP and estradiol, plus 24 h IVM) and control IVM (24 h) were compared. Biphasic IVM increased intra-oocyte glutathione and decreased ROS, up-regulated DNA-methyltransferase 1 and pentraxin 3 expression and led to an increase in rate of blastocyst development compared to control group (30.2% vs 17.2%; P<0.05). In conclusion, a biphasic IVM, including a pre-IVM with CNP, maintains oocyte meiotic arrest for 6 h and enhances the embryo developmental competence of oocytes from juvenile goats.Sandra Soto-Heras, Irene Menéndez-Blanco, Maria-Gracia Catalá, Dolors Izquierdo, Jeremy G. Thompson, Maria-Teresa Parami

Should emergence models for Lolium rigidum be changed throughout climatic conditions? The case of Spain

Lolium rigidum Gaudin is a troublesome weed worldwide, and its distribution may increase due to climate change. It has become resistant to several herbicides and, therefore, its control is problematic. On the other hand, its seed biology, with little dormancy and short-term viability, can play a major role in reducing its seed bank, which would improve control of this weed. For this reason, knowledge of the emergence patterns of this species entails improvements for its management. In this work seeds from one population of L. rigidum, collected in Northeastern Spain, were distributed and sown in 11 sites across Spain, and its emergence was followed every 2–7 days for two growing seasons (2016-17 and 2017–18). These emergence sequences were compared with previously published thermal time and hydrothermal time based models, and new models were developed afterwards. The hydrothermal time based model did not significantly improve models based solely on thermal time. The accuracy of these newly developed models varied from site to site, but in general, they worked better for Northern locations than for Southern locations. Thus, the experimental sites were split between North and South, and specific models for each region were developed. The model developed for Northern sites worked very well, but the one for Southern sites needs improvement. This works reveals that, with regard to this weed species, probably different models should be developed for different climatic conditions. The model developed with data from Northern sites should be validated with local populations, while the one developed with data from Southern sites will probably need to be revised or newly developed with data from more Southern populations.The group from the University of Lleida-Agrotecnio would also like to thank the Spanish Goverment for partial funding, through project AGL2017-83325-C4-2-R (AEI/FEDER/UE)

Modeling the emergence of North African Knapweed (Centaurea diluta), an increasingly troublesome weed in Spain

North African knapweed (Centaurea diluta Aiton) is an annual weed that is widespread in southern Spain and is of increasing concern in dryland cropping systems. Despite its expanding range in Spain, there is limited information on the emergence timing and pattern of this species, which is critical for developing more timely and effective management strategies. Therefore, there is a need to develop simple and reliable models to predict the timing and emergence of this annual weed under dryland conditions. A multi-location field experiment was established across Spain in 2016-2017 to assess the emergence of C. diluta. At each of 11 locations, seeds were sown in the fall, and emergence was recorded. Overall emergence averaged 39% in the first year across all sites and 11% in the second year. In both years, the main emergence flush occurred at beginning of the growing season. The three-parameter Weibull function best described seedling emergence of C. diluta. Emergence models were developed based on thermal time (TT) and hydrothermal time (HTT) and showed high predictability, as evidenced by root mean square error prediction (RMSEP) values of 10.8 and 10.7, respectively. The three cardinal points were established for TT and HHT at 0.5 ºC, 10 ºC and 35 ºC for base, optimal and ceiling temperature, while base water potential was estimated at -0.5MPa.We would like to thank Cátedra Adama and the Spanish Weed Science Society (SEMh) for providing financial support for this project. We are grateful to the many students and technicians who assisted with field work at the different experimental locations. We would like to thank the Spanish Goverment for partial funding, through project AVA 2019.020 "Advances in Technological Research in Winter Cereals: Genetic Improvement and Sustainable Management" financed with FEDER funds (80%). Also, Joel Torra acknowledges support from the Spanish Ministry of Science, Innovation and Universities (grant Ramon y Cajal RYC2018-023866-I)

A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis