Cannabinoid CB₂ Receptor Modulation by the Transcription Factor NRF2 is Specific in Microglial Cells

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor that has neuroprotective and anti-inflammatory effects, regulating more than 250 genes. As NRF2, cannabinoid receptor type 2 (CB2) is also implicated in the preservation of neurons against glia-driven inflammation. To this concern, little is known about the regulation pathways implicated in CB2 receptor expression. In this study, we analyze whether NRF2 could modulate the transcription of CB2 in neuronal and microglial cells. Bioinformatics analysis revealed an antioxidant response element in the promoter sequence of the CB2 receptor gene. Further analysis by chemical and genetic manipulations of this transcription factor demonstrated that NRF2 is not able to modulate the expression of CB2 in neurons. On the other hand, at the level of microglia, the expression of CB2 is NRF2-dependent. These results are related to the differential levels of expression of both genes regarding the brain cell type. Since modulation of CB2 receptor signaling may represent a promising therapeutic target with minimal psychotropic effects that can be used to modulate endocannabinoid-based therapeutic approaches and to reduce neurodegeneration, our findings will contribute to disclose the potential of CB2 as a novel target for treating different pathologies.This work was supported by the Spanish Ministry of Economy and Competitiveness (Grants Refs. SAF2016-76520-R to ILB and BFU2016-75973-R to MDG)

Conformationally restricted PACAP27 analogues incorporating type II/II′ IBTM β-Turn mimetics. Synthesis, NMR structure determination, and binding affinity

To probe the importance of a proposed β-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II′ β-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to 1H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM10,11]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an α-helical conformation within segment T7–L27. For residues S9–R12, our data seem more compatible with a segment of the α-helix than with the β-turn previously proposed for this fragment. In compound 1 the α-helix, also spanning T7–L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.Work at the Instituto de Quı́mica Médica and Universidad de Navarra was supported by CICYT (SAF 97 0030 and SAF 2000-0147), Fundación La Caixa (97/022) and Comunidad Autónoma de Madrid (08.5/0006/1998). Work at the Instituto de Estructura de la Materia was supported by DGICYT (PB98-0677) and the European Union (CEE B104-97-2086). Work at the Universidad de Barcelona was supported by Generalitat de Catalunya (CERBA). C.M.S. and M.M.-M. are recipients of a pre-doctoral and a post-doctoral fellowship, respectively, from the Comunidad Autónoma de Madrid, Spain. E.de O. is a post-doctoral fellow of Fundació Bosch i Gimpera, Universitat de Barcelona, Spain

Beneficial effect of systemic allogeneic adipose derived mesenchymal cells on the clinical, inflammatory and immunologic status of a patient with recessive dystrophic epidermolysis bullosa: A case report

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable inherited mucocutaneous fragility disorder characterized by recurrent blisters, erosions, and wounds. Continuous blistering triggers overlapping cycles of never-ending healing and scarring commonly evolving to chronic systemic inflammation and fibrosis. The systemic treatment with allogeneic mesenchymal cells (MSC) from bone marrow has previously shown benefits in RDEB. MSC from adipose tissue (ADMSC) are easier to isolate. This is the first report on the use of systemic allogeneic ADMSC, correlating the clinical, inflammatory, and immunologic outcomes in RDEB indicating long-lasting benefits. We present the case of an RDEB patient harboring heterozygous biallelic COL7A1 gene mutations and with a diminished expression of C7. The patient presented with long-lasting refractory and painful oral ulcers distressing her quality of life. Histamine receptor antagonists, opioid analgesics, proton-pump inhibitors, and low-dose tricyclic antidepressants barely improved gastric symptoms, pain, and pruritus. Concomitantly, allogeneic ADMSC were provided as three separate intravenous injections of 106 cells/kg every 21 days. ADMSC treatment was well-tolerated. Improvements in wound healing, itch, pain and quality of life were observed, maximally at 6-9 months post-treatment, with the relief of symptoms still noticeable for up to 2 years. Remarkably, significant modifications in PBL participating in both the innate and adaptive responses, alongside regulation of levels of profibrotic factors, MCP-1/CCL2 and TGF-beta, correlated with the health improvement. This treatment might represent an alternative for non-responding patients to conventional management. It seems critical to elucidate the paracrine modulation of the immune system by MSC for their rational use in regenerative/immunoregulatory therapies.This study was supported by a donation from Berritxuak-Elkartea (2015/00397/002), a collaborative rare disease association and, from La Paz University Hospital as well as by grants from the Community of Madrid (AvanCell-CM S2017/BMD-3692) and the Spanish Ministry of Economy and Competitiveness (SAF2017-86810-R). The UCMteamis supported by grants from the Spanish Institute of Health Carlos III (RD16/0011/0002) and the Spanish Ministry of Economy and Competitiveness (RTI2018-093899-B-I00). MJE is recipient of a contract funded by DEBRA-Spain

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.Acknowledgements: We thank Petros Tyrakis and Iván Martínez-Forero for critical reading and editing of the manuscript. Support was provided by the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the SPRI I+D COVID-19 fund (Gobierno Vasco). Personal fellowships: A.A.-V. (La Caixa Inphinit LCF/BQ/DR20/11790022), A.B. (AECC Bizkaia), A.G.d.R (Bikaintek), A.P. (Ramón y Cajal), B.J.-L. (Gob. Vasco), and E.P.-F. (Juan de la Cierva-Formación). M.L.M.-C. acknowledges RTC2019-007125-1, DTS20/00138, SAF2017-87301-R, and BBVA UMBRELLA project. M.L.-H. acknowledges the ISCIII for grant COV20-0170 and the Government of Cantabria for grant 2020UIC22-PUB-0019. O.M., J.-M.M., and N.G.A.A. acknowledge the Agencia Estatal de Investigación (Spain) for grants CTQ2015-68756-R, RTI2018-101269-BI00, and RTI2018-095700-B-I00, respectively. A.P. has received grant funding from the European Research Council (ERC), grant agreement number 804236 (Horizon 2020), and the FERO Foundation

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17 h of incubation, respectively. Interestingly, FTIR spectroscopy, 13C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.Financial support from the BIORENEW EU-project (NMP2-CT-2006-26456), Austrian Academic Exchange Programme (OEAD) and the Spanish projects BIO2007-28720-E, BIO2008-01533, and AGL2008-00709 is acknowledged

Polymerisation of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility

Prasetyo, Endry Nugroho et al.--The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17 h of incubation, respectively. Interestingly, FTIR spectroscopy, 13C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.Financial support from the BIORENEW EU-project (NMP2-CT-2006-26456), Austrian Academic Exchange Programme (ÖEAD) and the Spanish projects BIO2007-28720-E, BIO2008-01533, and AGL2008-00709 is acknowledged.Peer reviewe

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

Checklist of the vascular plants of the Cantabrian Mountains

We present the first standardized list of the vascular flora of the Cantabrian Mountains, a transitional zone between the Eurosiberian and Mediterranean biogeographic regions in northwestern Spain. The study area comprises 15000 km2 divided in UTM grid cells of 10 km x 10 km, for which we revised occurrence data reported in the Spanish Plant Information System (Anthos) and the online database of Iberian and Macaronesian Vegetation (SIVIM). We used a semi-automatic procedure to standardize taxonomic concepts into a single list of names, which was further updated by expert-based revision with the support of national and regional literature. In the current version, the checklist of the Cantabrian Mountains contains 2338 native species and subspecies, from which 56 are endemic to the study area. The nomenclature of the checklist follows Euro+Med in 97% of taxa, including annotations when other criteria has been used and for taxa with uncertain status. We also provide a list of 492 non-native taxa that were erroneously reported in the study area, a list of local apomictic taxa, a phylogenetic tree linked to The Plant List, a standardized calculation of Ellenberg Ecological Indicator Values for 80% of the flora, and information about life forms, IUCN threat categories and legal protection status. Our review demonstrates how the Cantabrian mountains represent a key floristic region in southern Europe and a relevant phytogeographical hub in south-western Europe. The checklist and all related information are freely accessible in a digital repository for further uses in basic and applied researchThis research was supported by the Jardín Botánico Atlántico de Gijón (SV-20-GIJON-JBA) and SEEDALP project (Spanish Reearch Agency; PID2019-108636GA/AEI/10.13039/501100011033)Peer reviewe

Influence of the Cumulative Incidence of COVID-19 Cases on the Mental Health of the Spanish Out-of-Hospital Professionals

This study aimed to analyze the psychological affectation of health professionals (HPs) of Spanish Emergency Medical Services (EMSs) according to the cumulative incidence (CI) of COVID19 cases in the regions in which they worked. A cross-sectional descriptive study was designed, including all HPs working in any EMS of the Spanish geography between 1 February 2021 and 30 April 2021. Their level of stress, anxiety and depression (DASS-21) and the perception of self-efficacy (GSES) were the study’s main results. A 2-factor analysis of covariance was used to determine if the CI regions of COVID-19 cases determined the psychological impact on each of the studied variables. A total of 1710 HPs were included. A third presented psychological impairment classified as severe. The interaction of CI regions with the studied variables did not influence their levels of stress, anxiety, depression or self-efficacy. Women, younger HPs or those with less EMS work experience, emergency medical technicians (EMT), workers who had to modify their working conditions or those who lived with minors or dependents suffered a greater impact from the COVID-19 pandemic in certain regions. These HPs have shown high levels of stress, anxiety, depression and medium levels of self-efficacy, with similar data in the different geographical areas. Psychological support is essential to mitigate their suffering and teach them to react to adverse events.This research was funded by Fundación ASISA and Sociedad Española de Urgencias y Emergencias (SEMES)