Living myocardial slices for the study of nucleic acid-based therapies

Gene therapy based on viral vectors offers great potential for the study and the treatment of cardiac diseases. Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac tissue from failing (dilated cardiomyopathy) hearts was also used to validate the model in a more translational setting. LMS were cultured at physiological sarcomere length for 72-h under electrical stimulation. Two recombinant AAV serotypes (AAV6 and AAV9) at different multiplicity of infection (MOI) expressing enhanced green fluorescent protein (eGFP) were added to the surface of rat LMS. AAV6 at 20,000 MOI proved to be the most suitable serotype without affecting LMS contractility or kinetics and showing high transduction and penetrability efficiency in rat LMS. This serotype exhibited 40% of transduction efficiency in cardiomyocytes and stromal cells while 20% of the endothelial cells were transduced. With great translational relevance, this protocol introduces the use of LMS as a model for nucleic acid-based therapies, allowing the acceleration of preclinical studies for cardiac diseases

Melanocortin receptor agonist NDP-α-MSH improves cognitive deficits and microgliosis but not amyloidosis in advanced stages of AD progression in 5XFAD and 3xTg mice

Introduction: Alzheimer's disease (AD) is the most frequent cause of dementia and still lacks effective therapy. Clinical signs of AD include low levels of endogenous melanocortins (MCs) and previous studies have shown that treatment with MC analogs induces neuroprotection in the early stages of AD.Methods: We investigated the neuroprotective role of MCs in two transgenic mouse models of severe AD using 5 and 7 month-old (mo) 5XFAD mice and 9 and 12 mo 3xTg mice. These mice were subjected to a chronic stimulation of MC receptors (MCRs) with MC analogue Nle4-D-Phe7-alpha-melanocyte stimulating hormone (NDP-alpha-MSH, 340 mu g/kg, i.p.). Mouse behavior and ex-vivo histological and biochemical analyses were performed after 50 days of treatment.Results:Our analysis demonstrated an improvement in cognitive abilities of AD mice at late stage of AD progression. We also showed that these protective effects are associated with decreased levels of hyperphosphorylated Tau but not with A beta burden, that was unaffected in the hippocampus and in the cortex of AD mice. In addition, an age-dependent NDP effect on glial reactivity was observed only in 3xTg mice whereas a global downregulation of p38 mitogen-activated protein kinase was selectively observed in 7 mo 5XFAD and 14 mo 3xTg mice.Conclusion: Our results suggest that MCR stimulation by NDP-alpha-MSH could represent a promising therapeutic strategy in managing cognitive decline also at late stage of AD, whereas the effects on neuroinflammation may be restricted to specific stages of AD progression

Identification of key receptor residues discriminating human chorionic gonadotropin (Hcg)-and luteinizing hormone (lh)-specific signaling

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gon-adotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying “murinizing” mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated ki-nases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone β-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken to-gether, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding

Avaliação da atividade externa e da coleta de pólen de abelhas-sem-ferrão (Meliponini) na meliponicultura integrada à produção agroecológica.

Resumo: A meliponicultura é uma atividade produtiva que envolve a criação de abelhas-sem-ferrão (ASF) a qual possui grande potencial de integração aos sistemas agroecológicos de produção. Essa integração é favorecida pela diversidade de plantas que disponibilizam recursos diversificados para as abelhas e pelo não uso de agrotóxicos. Entretanto, ainda há pouco conhecimento sobre a adaptação e o desenvolvimento de colônias de diferentes espécies de ASF na integração desses dois sistemas de produção. Esse trabalho teve por objetivo avaliar a atividade externa de ASF como um parâmetro do desenvolvimento das colônias e testar a hipótese que o fluxo de pólen em colônias da mesma espécie pode apresentar diferenças maiores durante a estação seca que na chuvosa devido ao efeito da sazonalidade na fenologia das plantas. Para isso, foram utilizadas colônias de três espécies de ASF existentes em dois meliponários integrados em duas áreas de produção agroecológica, denominadas Unidades de Aprendizagem (UA). Durante doze meses, duas colônias de cada espécie tiveram a atividade externa avaliadas semanalmente na UA em Mogi Mirim (SP) e por seis meses na UA em Nova Odessa (SP). A análise dos dados revelou que o efeito da sazonalidade na fenologia das plantas pode resultar em diferenças no fluxo de pólen entre colônias T. angustula e entre colônias de P. droryana. As diferenças encontradas no fluxo de pólen entre colônias de mesma espécie podem ainda indicar um desenvolvimento diferencial desses ninhos; contudo, outros parâmetros devem ser considerados para melhor qualificar as condições coloniais. Abstract: Meliponiculture is a productive activity that involves the rearing of stingless bees (ASF) and has great potential for integration into agroecological production systems. This integration is favored by the diversity of plants, which provide diversified resources for bees, and by the absence of pesticide use. However, there is still little knowledge about the adaptation and development of colonies of different ASF species in the integration of these two production systems. This study aimed to evaluate external activity as a parameter for colony development and test the hypothesis that the pollen flow in colonies of the same species may present greater differences during the dry season than in the rainy season due to the effect of seasonality on plant phenology. For this purpose, colonies of three ASF species were used in two meliponaries integrated into two agroecological production areas, referred to as Learning Units (UA). For twelve months, two colonies of each species had their external activity evaluated weekly at the AU in Mogi Mirim (SP) and for six months at the AU in Nova Odessa (SP). Data analysis revealed that the effect of seasonality on plant phenology may result in differences in pollen flow between T. angustula colonies and between P. droryana colonies. Differences found in pollen flow between colonies of the same species may also indicate a differential development of these nests; however, other parameters must be considered to better qualify colonial conditions.CIIC 2023. Nº 23411

One year after on Tyrrhenian coasts: The ban of cotton buds does not reduce their dominance in beach litter composition

In January 2019, Italy banned the sale of plastic cotton buds, which is one of the most abundant litter items entering the sea and then washing ashore. However, since the ban came into force, no studies have been carried out to assess whether the measure has actually led to the reduction of plastic cotton buds accumulating on Italian coasts. Here we aim at evaluating the effectiveness of the ban in reducing the amount of cotton buds reaching sandy beaches of the Tyrrhenian coast. Specifically, we monitored the accumulation of beach litter for one year since the ban came into force. By surveying eight coastal sites from winter 2019 to winter 2020, we collected a total of 52,824 items mostly constituted by plastic debris (97.6%). We found that cotton buds were the most abundant item (42.3% of total litter), followed by plastic (28.5%) and polystyrene (5.43%) fragments. Our preliminary assessment suggests that the ban has so far not led to a sensible reduction in the amount of cotton buds entering the marine ecosystem. This was to be expected since implementation strategies are still lacking (i.e. no economic sanctions can be imposed in case of non-compliance) and bans are differently implemented among countries facing the Mediterranean Sea, calling for law enforcement and implementation at the national and international levels

Nutritive value of rations containing sugar cane, poultry litter and corn

Realizou-se, no campus da Faculdade de Ciências Agrárias e Veterinárias da UNESP, em Jaboticabal, SP, um estudo com o objetivo de determinar a ingestão voluntária e a digestibilidade aparente de rações contendo diferentes relações de cana-de-açúcar: cama-de-frango (CC1 = 65,50 : 34,50%; CC2 = 58,00 : 42,00%; e CC3 - 52,00 : 48,00% combinadas com três níveis de milho (M1 = 0 kg; M2 = 0,75 kg e M3 = 1,50 kg), através do delineamento em blocos casualizados em esquema fatorial 3 com três repetições. Utilizaram-se nove bezerros pesando, em média, 334 kg. Através da análise de regressão, detectou-se efeito linear positivo (P < 0,01) entre os níveis de milho e a digestibilidade aparente da matéria seca, da matéria orgânica, do extrato etéreo, do extrato não nitrogenado e dos teores de nutrientes digestíveis totais. As proporções de cana: cama-de-frango com menores quantidades de cana proporcionaram aumentos na digestibilidade da proteína e da fibra bruta (P < 0,01). Não houve interação significativa entre os níveis de milho e as proporções de cana: cama-de-frango. O valor nutritivo e a ingestão de matéria seca total foram significativamente aumentadas nas rações com os níveis crescentes de milho (P < 0,01).This work was conduced at Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal-UNESP, São Paulo, Brazil, with the objective of finding out the intake and apparent digestibility of the nutrients in rations with different proportions of sugar cane (SC) and poultry litter (PL): 65.50 : 34.50%; 58.00 : 42.00% and 52.00 : 48.00% combined with three levels of corn: 0; 0.15 kg an 1,50 kg. Treatments were studied according to a completely randomized block design 32 with three replications. Nine steers with an initial average weight of 334 kg were used. Linear regression studies showed significant effects (P < 0.01) of the different levels of corn on apparent digestibility of the dry matter, organic matter, ether extract, nitrogen free extract and total digestible nutrients. Proportions of SC/PL with lower sugarcane ration increased crude fiber and protein digestibility (P < 0.01). There was no significant interaction of the levels of corn and different proportions of sugarcane to poultry litter. Nutritive value and total intake of dry matter increased with the levels of corn in the ration (P < 0.01)