Suchergebnisse

The development of physiological models that reproduce SARS-CoV-2 infection in primary human cells will be instrumental to identify host-pathogen interactions and potential therapeutics. Here, using cell suspensions directly from primary human lung tissues (HLT), we have developed a rapid platform for the identification of viral targets and the expression of viral entry factors, as well as for the screening of viral entry inhibitors and anti-inflammatory compounds. The direct use of HLT cells, without long-term cell culture and in vitro differentiation approaches, preserves main immune and structural cell populations, including the most susceptible cell targets for SARS-CoV-2; alveolar type II (AT-II) cells, while maintaining the expression of proteins involved in viral infection, such as ACE2, TMPRSS2, CD147 and AXL. Further, antiviral testing of 39 drug candidates reveals a highly reproducible method, suitable for different SARS-CoV-2 variants, and provides the identification of new compounds missed by conventional systems, such as VeroE6. Using this method, we also show that interferons do not modulate ACE2 expression, and that stimulation of local inflammatory responses can be modulated by different compounds with antiviral activity. Overall, we present a relevant and rapid method for the study of SARS-CoV-2. ; This work was primarily supported by a grant from the Health Department of the Government of Catalonia (DGRIS 1_5). This work was additionally supported in part by the Spanish Health Institute Carlos III (ISCIII, PI17/01470; PI19CIII/00004; PI21CIII/00025 and COV20-00679 (MPY 222-20)), the Spanish Secretariat of Science and Innovation and FEDER funds (grant RTI2018-101082-B-I00 [MINECO/FEDER]), the Spanish AIDS network Red Temática Cooperativa de Investigación en SIDA (RD16/0025/0007 and RD16CIII/0002/0001), the European Regional Development Fund (ERDF), the Fundació La Marató TV3 (grants 201805-10FMTV3 and 201814- 10FMTV3), the Gilead fellowships GLD19/00084 and GLD18/00008 and the Becas Taller Argal 2020. M.J.B is supported by the Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). N.M. is supported by a Ph.D. fellowship from the Vall d'Hebron Institut de Recerca (VHIR). The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript. ; No

This work was supported by grants CTQ2014-55474-C2-1-R, CTQ2014-55474-C2-2-R and CTQ2017-86125-P from the Ministerio Economia, Industria y Competitividad (co-financed by FEDER funds). SP is supported by a FPU fellowship (FPU17/ 04749). We acknowledge the University of Granada (Spain) cell culture, animal and microscopy central facilities (CIC-UGR). ; The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due to their smaller size, low immunogenicity, and low-cost production. Although covalent strategies for the preparation of such ScFv-based therapeutic conjugates are prevalent, this approach is not straightforward, as it requires prior chemical activation and/or modification of both the ScFv and the therapeutics for the application of robust chemistries. A non-covalent alternative based on ScFv fused to maltose-binding protein (MBP) acting as a binding adapter is proposed for active targeted delivery. MBP-ScFv proves to be a valuable modular platform to synergistically bind maltose-derivatized therapeutic cargos through the MBP, while preserving the targeting competences provided by the ScFv. The methodology has been tested by using a mutated maltose-binding protein (MBP I334W) with an enhanced affinity toward maltose and an ScFv coding sequence toward the human epidermal growth factor receptor 2 (HER2). Non-covalent binding complexes of the resulting MBP-ScFv fusion protein with diverse maltosylated therapeutic cargos (a near-infrared dye, a maltosylated supramolecular beta-cyclodextrin container for doxorubicin, and non-viral polyplex gene vector) were easily prepared and characterized. In vitro and in vivo assays using cell lines that express or not the HER2 epitope, and mice xenografts of HER2 expressing cells demonstrated the capability and versatility of MBP-ScFv for diagnosis, imaging, and drug and plasmid active targeted tumor delivery. Remarkably, the modularity of the MBP-ScFv platform allows the flexible interchange of both the cargos and the coding sequence for the ScFv, allowing ad hoc solutions in targeting delivery without any further optimization since the MBP acts as a pivotal element. ; Ministerio Economia, Industria y Competitividad - FEDER funds CTQ2014-55474-C2-1-R CTQ2014-55474-C2-2-R CTQ2017-86125-P ; Spanish Government FPU17/04749

[Abstract] Knowledge and research results about hand osteoarthritis (hOA) are limited due to the lack of samples and animal models of the disease. Here, we report the generation of two induced pluripotent stem cell (iPSC)-lines from patients with radiographic hOA. Furthermore, we wondered whether these iPSC-lines carried single nucleotide polymorphisms (SNPs) within genes that have been associated with hOA. Finally, we performed chondrogenic differentiation of the iPSCs in order to prove their usefulness as cellular models of the disease. We performed a non-integrative reprogramming of dermal fibroblasts obtained from two patients with radiographic rhizarthrosis and non-erosive hOA by introducing the transcriptional factors Oct4, Sox2, Klf4 and c-Myc using Sendai virus. After reprogramming, embryonic stem cell-like colonies emerged in culture, which fulfilled all the criteria to be considered iPSCs. Both iPSC-lines carried variants associated with hOA in the four studied genes and showed differences in their chondrogenic capacity when compared with a healthy control iPSC-line. To our knowledge this is the first time that the generation of iPSC-lines from patients with rhizarthrosis and non-erosive hOA is reported. The obtained iPSC-lines might enable us to model the disease in vitro, and to deeper study both the molecular and cellular mechanisms underlying hOA. ; This study was carried out thanks to the funding from Fundación Española de Reumatología (Proyectos 2014), Proyectos de Investigación 2016 (PI16/02124) and 2017 (PI17/02197) from Instituto de Salud Carlos III-General Subdirection of Assesment and Promotion of the Research – European Regional Development Fund (FEDER) "A way of making Europe", Rede Galega de Terapia Celular and Grupos con Potencial de Crecemento, Xunta de Galicia (R2016/036, R2014/050, CN2012/142 and GPC2014/048); Deputación da Coruña (BINV-CS/2015); University of A Coruña; Centro de Investigación Biomédica en Red-Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN). Rocío Castro-Viñuelas, María Piñeiro-Ramil and Silvia Rodríguez-Fernández are granted by a predoctoral fellowship from Xunta de Galicia and European Union (European Social Fund) and Clara Sanjurjo-Rodríguez is beneficiary of a postdoctoral fellowship from Xunta de Galicia ; Xunta de Galicia; R2016/036 ; Deputación da Coruña; BINV-CS/2015 ; Xunta de Galicia; R2014/050 ; Xunta de Galicia; CN2012/142 ; Xunta de Galicia; GPC2014/048

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment. ; S.R.P.'s laboratory is partially funded by funds from the Spanish National Research and Development Plan, Instituto de Salud Carlos III and FEDER [PI17/02303 to S.R.P]; R.T.R. is supported by a fellowship from the AECC scientific foundation; J.M.B. is supported by Spanish Ministry of Education, Culture and Sport [FPU15/01978]; J.B.'s laboratory is partially funded by FEDER funds, H2020 BRIDGES project and the Spanish Network on Rare Diseases (CIBERER) [FIS PI16/00440]; Faculty of Medicine at the Norwegian University of Science and Technology and the Central Norway Regional Health Authority supports [46056921 to A.S. and H.E.K.]; Svanhild and Arne Must's Fund for Medical Research (to A.S. and H.E.K.); Norwegian Research Council (to T.V.); SIN-TEF SEP project 102020885 (to T.V.); Vinnova (to A.C.K and P.S.); the Torsten and Ragnar Soderberg Foundation (to T.H.) and the Helleday Foundation (to C.B.). Funding for open access charge and project support: European Union's Horizon 2020 research and innovation programunder the Marie Sklodowska-Curie grant agreement [722729 to A.P., B.M.F.H., T.H.]; European Research Council [ERC TAROX-695376 to T.H.]; Swedish Research Council (to T.H. and P.S.); Swedish Cancer Society (to T.H. and P.S.); Swedish Children's Cancer Foundation (to T.H.); Swedish Pain Relief Foundation (to T.H.). ; Sí

Most neurodegenerative diseases are characterized by a complex and mostly still unresolved pathology. This fact, together with the lack of reliable models, have precluded the development of effective therapies counteracting the disease progression. In the past few years, several studies have evidenced that lack of proper functionality of glial cells (astrocytes, microglia and oligodendrocytes) has a key role in the pathology of several neurodegenerative conditions including Alzheimer´s disease, amyotrophic lateral sclerosis and multiple sclerosis among others. However, this glial dysfunction is poorly modelled by available animal models, and we hypothesize that patientderived cells can serve as a better platform where to study this glial dysfunction. In this sense, human pluripotent stem cells (hPSCs) has revolutionized the field allowing the generation of disease-relevant neural cell types that can be used for disease modelling, drug screening and, possibly, cell transplantation purposes. In the case of the generation of oligodendrocytes (OLs) from hPSCs, we have developed a fast and robust protocol to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The generated cells resemble primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OLneuron co-cultures, effective myelination of neurons can also be demonstrated. This platform is being translated as well to the generation of the other glial cell types, allowing the derivation of patient-specific glial cells where to model disease-specific dysfunction. This methodology can be used for elucidating pathogenic pathways associated with neurodegeneration and to identify therapeutic targets susceptible of drug modulation, contributing to the development of novel and effective drugs for these devastating disorders. ; Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Supported by PI18/01557 (to AG) and P18/1556 (to JV) grants from ISCiii of Spain co-financed by FEDER funds from European Union, and PI-0276-2018 grant (to JAGL) from Consejeria de Salud of Junta de Andalucia. JAGL held a postdoctoral contract from the I Research Plan Propio of the University of Malaga. CV and KE were supported by IWT-SBO-150031-iPSCAF and the Thierry Lathran Foundation grant – ALS-OL, and KN by FWO1166518N

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO2NPs, SiO2NPs, Fe3O4NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe3O4NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (>700 μg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 μg/ml) concentration. Fe3O4NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO2NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake. ; This article was created by the realization of the H2020 project HISENTS No. 685817, COST Action CA15132, VEGA grant 2/0056/17, project "Center of excellence of environmental health", ITMS No. 26240120033, based on the support of operational Research and development program financed from the European Regional Development Fund; and the EEA projectSK0020. Filip Razga received support within the SASPRO Programme (Project No. 0057/01/02) co-funded by the European Union and the Slovak Academy of Sciences. ; Peer reviewed

Mild traumatic brain injury (mTBI) disproportionately affects military service members and is very difficult to diagnose. To-date, there is currently no blood-based, diagnostic biomarker for mTBI cases with persistent post concussive symptoms. To examine the potential of neuronally-derived (NDE) and astrocytic-derived (ADE) exosome cargo proteins as biomarkers of chronic mTBI in younger adults, we examined plasma exosomes from a prospective longitudinal study of combat-related risk and resilience, marine resiliency study II (MRSII). After return from a combat-deployment participants were interviewed to assess TBI exposure while on deployment. Plasma exosomes from military service members with mTBI (mean age, 21.7 years, n = 19, avg. days since injury 151), and age-matched, controls (deployed service members who did not endorse a deployment-related TBI or a pre-deployment history of TBI; mean age, 21.95 years, n = 20) were precipitated and enriched against a neuronal adhesion protein, L1-CAM, and an astrocyte marker, glutamine aspartate transporter (GLAST) using magnetic beads to immunocapture the proteins and subsequently selected by fluorescent activated cell sorting (FACS). Extracted protein cargo from NDE and ADE preparations were quantified for protein levels implicated in TBI neuropathology by standard ELISAs and on the ultra-sensitive single molecule assay (Simoa) platform. Plasma NDE and ADE levels of Aβ42 were significantly higher while plasma NDE and ADE levels of the postsynaptic protein, neurogranin (NRGN) were significantly lower in participants endorsing mTBI exposure compared to controls with no TBI history. Plasma NDE and ADE levels of Aβ40, total tau, and neurofilament light (NFL), P-T181-tau, P-S396-tau were either undetectable or not significantly different between the two groups. In an effort to understand the pathogenetic potential of NDE and ADE cargo proteins, neuron-like cultures were treated with NDE and ADE preparations from TBI and non-TBI groups. Lastly, we determined that plasma NDE but not ADE cargo proteins from mTBI samples were found to be toxic to neuron-like recipient cells in vitro. These data support the presence of markers of neurodegeneration in NDEs of mTBI and suggest that these NDEs can be used as tools to identify pathogenic mechanisms of TBI.

Mild traumatic brain injury (mTBI) disproportionately affects military service members and is very difficult to diagnose. To-date, there is currently no blood-based, diagnostic biomarker for mTBI cases with persistent post concussive symptoms. To examine the potential of neuronally-derived (NDE) and astrocytic-derived (ADE) exosome cargo proteins as biomarkers of chronic mTBI in younger adults, we examined plasma exosomes from a prospective longitudinal study of combat-related risk and resilience, marine resiliency study II (MRSII). After return from a combat-deployment participants were interviewed to assess TBI exposure while on deployment. Plasma exosomes from military service members with mTBI (mean age, 21.7 years, n = 19, avg. days since injury 151), and age-matched, controls (deployed service members who did not endorse a deployment-related TBI or a pre-deployment history of TBI; mean age, 21.95 years, n = 20) were precipitated and enriched against a neuronal adhesion protein, L1-CAM, and an astrocyte marker, glutamine aspartate transporter (GLAST) using magnetic beads to immunocapture the proteins and subsequently selected by fluorescent activated cell sorting (FACS). Extracted protein cargo from NDE and ADE preparations were quantified for protein levels implicated in TBI neuropathology by standard ELISAs and on the ultra-sensitive single molecule assay (Simoa) platform. Plasma NDE and ADE levels of Aβ42 were significantly higher while plasma NDE and ADE levels of the postsynaptic protein, neurogranin (NRGN) were significantly lower in participants endorsing mTBI exposure compared to controls with no TBI history. Plasma NDE and ADE levels of Aβ40, total tau, and neurofilament light (NFL), P-T181-tau, P-S396-tau were either undetectable or not significantly different between the two groups. In an effort to understand the pathogenetic potential of NDE and ADE cargo proteins, neuron-like cultures were treated with NDE and ADE preparations from TBI and non-TBI groups. Lastly, we determined that plasma NDE but not ADE cargo proteins from mTBI samples were found to be toxic to neuron-like recipient cells in vitro. These data support the presence of markers of neurodegeneration in NDEs of mTBI and suggest that these NDEs can be used as tools to identify pathogenic mechanisms of TBI.

HtrA2 (high-temperature requirement 2) is a human mitochondrial protease that has a role in apoptosis and Parkinson's disease. The structure of HtrA2 with an intact catalytic triad was determined, revealing a conformational change in the active site loops, involving mainly the regulatory LD loop, which resulted in burial of the catalytic serine relative to the previously reported structure of the proteolytically inactive mutant. Mutations in the loops surrounding the active site that significantly restricted their mobility, reduced proteolytic activity both in vitro and in cells, suggesting that regulation of HtrA2 activity cannot be explained by a simple transition to an activated conformational state with enhanced active site accessibility. Manipulation of solvent viscosity highlighted an unusual bi-phasic behavior of the enzymatic activity, which together with MD calculations supports the importance of motion in the regulation of the activity of HtrA2. HtrA2 is an unusually thermostable enzyme (TM=97.3 °C), a trait often associated with structural rigidity, not dynamic motion. We suggest that this thermostability functions to provide a stable scaffold for the observed loop motions, allowing them a relatively free conformational search within a rather restricted volume. ; We would like to thank Professor Margarida Bastos and Dr. Frederico Silva for assistance with calorimetry, Professor Manuel J.B. Marques with refractometry, Professor Eduardo Marques and Dr. João Neves with viscosometry, Dr. Trent Balius with hierarchical clustering, Professor Brian K. Shoichet with interpreting protein stability measurements, Dr. Hélder Maiato, Dr. Cristina Ferrás, Dr. Emma Commas-Casellas and Luísa Ferreira for help with cell culture. Funding to Biomolecular Structure and Function lab was provided by the project Norte-01-0145-FEDER-000008 – Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). CM, MJR and PAF acknowledge the financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. C.M. is a recipient of a FCT PhD fellowship (SFRH/BD/84016/2012). M.M. thanks the Fundação para a Ciência e a Tecnologia (FCT) for its support through the Ciência 2008 program. ; info:eu-repo/semantics/publishedVersion

Nanomaterials give rise to unique biological reactivity that needs to be thoroughly investigated. The quest for enhanced magnetic nanomaterials of different shapes, magnetic properties, or surface coatings continues for applications in drug delivery, targeting therapies, biosensing, and magnetic separation. In this context, the use of simple in vivo models, such as Caenorhabditis elegans, to biologically evaluate nanoparticles is currently in increasing demand as it offers low-cost and information-rich experiments. In this work, we evaluated how surface modification (citrate- and protein-coated) of superparamagnetic iron oxide nanoparticles (C-SPIONs and BSA-SPIONs, respectively) induces changes in their toxicological profile and biodistribution using the animal model C. elegans and combining techniques from materials science and biochemistry. The acute toxicity and nanoparticle distribution were assessed in two populations of worms (adults and larvae) treated with both types of SPIONs. After 24 h treatment, nanoparticles were localized in the alimentary system of C. elegans; acute toxicity was stronger in adults and larvae exposed to C-SPIONs rather than BSA-SPIONs. Adult uptake was similar for both SPION types, whereas uptake in larvae was dependent on the surface coating, being higher for BSA-SPIONs. Nanoparticle size was evaluated upon excretion, and a slight size decrease was found. Interestingly, all results indicate the protective effects of the BSA to prevent degradation of the nanoparticles and decrease acute toxicity to the worms, especially at high concentrations. We argue that this relevant information on the chemistry and toxicity of SPIONs in vivo could not be gathered using more classical in vitro approaches such as cell culture assays, thus endorsing the potential of C. elegans to assess nanomaterials at early stages of their synthetic formulations. ; C. elegans N2 and E. coli OP50 were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The research leading to these results has received funding from the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program (FP7/2007-2013) under REA grant agreement nº 303630 and cofounded by the European Social Fund. Authors acknowledge the funding from Spanish Ministry of Economy MAT 2012-35324 and FEDER funds, the Generalitat de Catalunya 2014SGR213, the COST Action MP1202, Ramon y Cajal grant RYC-2010-06082 (AL), China Scholarship Council fellowship (SMY, 201206150053), and FPU fellowship FPU12/05549 (LGM). ; Peer Reviewed

PURPOSE: The precise molecular cues required for photoreceptor development are still unknown. Pax6 and Crx are essential during early retinal development and for photoreceptor differentiation, respectively. The lipid molecule docosahexaenoic acid (DHA) has also been shown to promote photoreceptor differentiation. Pax6 expression during the early steps in photoreceptor development and whether the mutual contribution of Crx and DHA enhances photoreceptor differentiation were investigated. METHODS: Neuroblast proliferation, Crx, and Pax6 expression were investigated in rat retinas in vivo and in neuronal cultures with or without DHA. BrdU incorporation, nestin and opsin expression, apical differentiation, and axonal outgrowth were determined by phase microscopy and immunochemistry. RESULTS: Pax6 expression occurred in all proliferating retinal neuroblasts in vivo; however, after their last mitotic division, photoreceptors stopped expressing Pax6 and started expressing Crx. In vitro, photoreceptor progenitors also showed a switch from Pax6 to Crx expression immediately after they exited the cell cycle and started differentiation. In contrast, those progenitors differentiating into amacrine neurons continued expressing Pax6 and did not express Crx. Most postmitotic photoreceptors expressing Crx showed little axon development and few of them expressed opsin. The addition of DHA dramatically increased differentiation in Crx-positive photoreceptors, enhancing opsin expression, apical differentiation, and axonal outgrowth, without affecting Crx expression. CONCLUSIONS: The results suggest that Pax6 and Crx expression are mutually exclusive during photoreceptor differentiation. Onset of Crx expression may provide a permissive stage that is essential to initiate photoreceptor differentiation, but additional support of DHA, among other environmental signals, is necessary to accomplish further differentiation. ; Fil: Garelli, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina ; Fil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina ; Fil: Politi, Luis Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina

Mild traumatic brain injury (mTBI)/ concussion accounts for 70-90% of all reported TBI cases in the United States and can cause long-term neurological outcomes that negatively impact quality of life. Previous studies revealed that increased blood-brain barrier (BBB) leakage is correlated with poor neurological outcomes after mTBI, yet the biological mechanisms linking BBB damage to the onset of neurological deficits after mTBI are not well understood. Previously, we found that astrocytes lose expression of homeostatic proteins after mTBI, characterizing the changes in astrocytic protein expression as an "atypical astrocyte response." Yet, the upstream mechanisms that induce this atypical astrocyte response after mTBI have yet to be elucidated. In models of more severe TBI, exposure to blood-borne factors triggers astrogliosis via upregulation in markers, such as glial fibrillary acidic protein (GFAP), but how exposure to blood-borne factors affects astrocyte protein expression in the context of mTBI is not well understood. Therefore, we hypothesized that mTBI-induced BBB damage causes atypical astrocytes via exposure to blood-borne factors. To test this hypothesis, we use a mTBI mouse model, two-photon microscopy, an endothelial cell-specific genetic ablation model, and serum-free primary astrocyte cultures. Here, we found that mTBI causes BBB damage through the loss of proteins involved in maintaining the BBB's physical and metabolic barriers, and BBB damage is sustained long-term after injury. Also, we demonstrated that leakage of blood-borne factors is sufficient to trigger atypical astrocytes, and plasma exposure triggers a similar response in vitro. Overall, these findings suggest that mTBI induces long-term BBB damage, and exposure to blood-borne factors triggers the loss of key homeostatic astrocytic proteins involved in maintaining healthy neuronal function. ; Doctor of Philosophy ; Mild traumatic brain injury (mTBI)/ concussion makes up 70-90% of all TBI cases reported in the United States and is commonly observed after car crashes, sports-related tackles, and blast exposure during military combat. People who experience mTBI develop debilitating long-term neurological consequences, such as sleep disturbances, depression, and dementia. Clinical data suggests mTBI causes damage to the barrier between the brain and blood, known as the blood-brain barrier (BBB). This damage has been correlated to the onset of poor neurological deficits, yet how damage to this barrier is causally linked to long-term neurological consequences remains to be fully understood. In our lab, we found that mTBI causes loss of proteins important for maintaining a healthy environment in the brain in specialized cells called astrocytes. However, the biological events that trigger the loss of protein expression in astrocytes after mTBI have yet to be fully investigated. Thus, we hypothesized that mTBI causes loss of these proteins via leakage of blood-borne factors. To test this hypothesis, we used a mTBI mouse model, two-photon microscopy, genetic manipulation, and cell cultures. In our studies, we found that mTBI triggers BBB damage via loss of proteins that make up its protective properties. Also, we demonstrated that leakage of blood-borne factors is sufficient to cause loss of astrocyte-specific proteins both in brain and cell cultures. Altogether, we show that a single mTBI is sufficient to cause loss of astrocyte-specific protein expression via exposure to blood-borne factors. These findings may point to targeting either the blood-borne factor(s) or their corresponding receptor pathways in astrocytes to halt the progression of long-term neurological deficits after mTBI.

Chapter 1: Water decontamination through Thiamethoxam removal using DL menthol-octanoic acid Deep Eutectic Solvent : Molecular dynamics insights -- Chapter 2: Methyl Red dye abatement from aqueous solution using Calcium Ferrite and Manganese Ferrite magnetic nanocomposite: Kinetics and isotherm study -- Chapter 3: Adsorption of Fluoride onto PANI-Cl jute fibre - designing higher flow rate and initial concentration column reactor from a batch reactor -- Chapter 4: Biosynthesis of Nano Zero valent Iron (nZVI) using Shorea robusta Leaf Extract and its application in UV-assisted Photocatalytic Degradation of Methyl Orange -- Chapter 5: Method Development for detection of 2-Methylpyridine by High Performance Liquid Chromatography -- Part 2 - Industrial effluent treatment, reuse and conservation -- Chapter 6: Importance of cost functions for biological treatment of wastewater -- Chapter 7: Removal of heavy metals by Laterite soil -- Chapter 8: Removal of Methylene Blue from wastewater by Red Sandy soil-Based Alkali activated binder -- Chapter 9: Assessment and Treatment of iron from industrial wastewater using activated carbon derived from Parkia Speciosa pod -- Chapter 10: Activated Carbon developed from Phumdi biomass and Deccan Hemp for adsorption of Methylene Blue -- Chapter 11: An experimental study of Metanil Yellow dye remediation using Fe-Mn Bimetal oxide composites -- Chapter 12: Optimization of electrocoagulation process parameters using magnesium electrodes for treating pharmaceutical wastewater containing salicylic acid -- Chapter 13: Optimization of process parameters for biodegradation of Cresol by mixed bacterial culture using Response Surface Methodology -- Chapter 14: Biological degradation of Cresol containing wastewater using mixed microbial culture in presence of heavy metals -- Part 3 - Monitoring of industrial emission and control -- Chapter 15: Development of a simplistic model for the prediction of reactive air pollutants in the atmosphere -- Chapter 16: Controlling Air and Metal pollution in industrial area Singrauli, India: Role of plants -- Part 4 - Industrial solid waste management -- Chapter 17: Enhancing the dewatering ability of sludge by locally available biomass -- Chapter 18: Production of Xylose from Water Hyacinth Biomass (WHB) (lignocellulosic waste) for Xylitol production: waste to wealth -- Chapter 19: A simplistic mathematical model for two-stage Anaerobic Digestion of plastic wastes -- Part 5 - Handling and disposal of hazardous waste -- Chapter 20: Modeling migration of Cr(VI) contaminant through Clay Liner using Hydrus-3d -- Chapter 21: Electrokinetic remediation of total Chromium from contaminated soil -- Chapter 22: Low-Cost recovery of Cadmium from wastewater by soil bacteria -- Part 6 - Case Studies on industrial pollution control -- Chapter 23: Tannery waste management in India: A case study -- Chapter 24: Removal of natural organics and selected antibiotics using PAC and AL electrode: A study for Jumar River -- Chapter 25: Quantification of floating plastics using UAV images and Identification of Microplastics in Ukkadam tank -- Chapter 26: Study of wastewater treatment in Hindustan Coca-Cola plant at Khurda -- Part 7 - Innovative technologies in industrial waste management -- Chapter 27: In Vitro Performance Analysis of Ti and Zn doped Hydroxyapatite made from waste Eggshells -- Chapter 28: Production of non-toxic, non-polluting herbal soaps using plant extracts having anti-microbial activity -- Chapter 29: Decolourization of Textile Dye RR 141 using electrochemical process.

La papa es uno de los alimentos más importantes a nivel mundial, especialmente para Suramérica donde se reporta su origen. Existen las papas nativas, las cuales se reconocen por su diversidad de texturas, colores y sabores. Este importante recurso genético ha sido conservado y cultivado por habitantes de la Zona Andina durante muchos años. En Colombia, las papas nativas son cultivadas y comercializadas principalmente en los departamentos de Cundinamarca, Boyacá y Nariño. Sin embargo, existen algunos limitantes que dificultan su posicionamiento a nivel comercial; dentro de los cuales se destacan el desconocimiento sobre su existencia, baja disponibilidad de semilla certificada, índices bajos de rendimiento y productividad, presencia de fitopatógenos por su propagación vegetativa, la falta de tecnología y herramientas que fortalezcan su producción y la inexistencia de programas locales enfocados a su investigación y asistencia técnica. Por lo anterior, se estableció una alianza triple hélice entre la universidad, empresa y Estado, con el fin de obtener materiales tradicionales de papa limpios, caracterizados con descriptores etnobotánicos, agronómicos, nutricionales y moleculares, enfocados hacia la obtención de individuos élite para su promoción agroeconómica. Esta caracterización se logró gracias a la implementación metodológica de dos líneas estratégicas, que se enmarcaron desde el trabajo continuo en laboratorio y en campo, mediante el establecimiento de parcelas experimentales, hasta la interacción con la comunidad para la divulgación y la apropación social del conocimiento. Como resultados se obtuvieron protocolos efectivos para lograr materiales limpios de genotipos ancestrales de papa, mediante la técnica de cultivo de tejidos in vitro . El material propagado fue examinado por medio de técnicas microbiológicas para la detección de hongos, bacterias y virus. Con lo anterior se aseguró la entrega de materiales limpios de papa nativa a los agricultores de los municipios de Ventaquemada y Chiscas, en el departamento de Boyacá. Adicionalmente, la micropropagación aseguró la caracterización molecular y la conservación in vitro de germoplasma de morfotipos nativos de papa del departamento. En el componente molecular, se logró establecer las relaciones genéticas entre los genotipos de papa nativa cultivadas en Boyacá. Esta información será base fundamental para el establecimiento de individuos potenciales parentales en programas de mejoramiento genético a nivel departamental. En el componente de campo se logró determinar el rendimiento de cada genotipo de papa bajo las condiciones de dos localidades, encontrando que algunos de ellos presentan mejor desarrollo y producción dependiendo de su adaptación al ambiente de la localidad evaluada. También se logró determinar los parámetros nutricionales de los materiales de papa nativos evaluados. Esta información le permitirá al agricultor impulsar la comercialización de productos en fresco e industrializados, con características organolépticas únicas en el mercado regional y nacional. El establecimiento de las siembras y la caracterización de los genotipos también crea un hito importante en la identificación morfológica de los materiales locales. La descripción botánica como la flor, la disposición de las hojas, el color del tallo, la coloración en el tubérculo y la forma de crecimiento, son caracteres universales que permitirán la diferenciación de estos materiales por parte de la comunidad. La apropiación social del conocimiento y la intervención de profesionales en las diferentes áreas y disciplinas, permitió el uso eficiente de la información, dando lugar a la generación de nuevos conocimientos de amplio impacto. Así mismo este libro de investigación muestra la experiencia exitosa de la vinculación de los actores sociales en la divulgación, generación y promoción de contenido científico, como herramienta de alto impacto en favor de la promoción y fomento de materiales nativos de papa. Es importante resaltar que, este libro cuenta con códigos QR a través de los cuales el lector podrá tener acceso directo al documental titulado: "Los Colores y Sabores de mi Tierra" , así como a tres cápsulas explicativas y complementarias a los contenidos del libro, integrando el estudio científico y los saberes tradicionales de las comunidades, propiciando la transferencia de tecnologías, en beneficio de los productores y del territorio. ; The potato is one of the most important foods worldwide, especially in South America where it originated. Within the potato genotypes, native potatoes are included, which are recognized for their diversity of textures, colors and flavors. This important genetic resource has been conserved and cultivated by the inhabitants of the Andean zone for many years. In Colombia, the native genotypes are cultivated and commercialized mainly in the departments of Cundinamarca, Boyacá and Nariño. However, there are some limitations that hinder its positioning at a commercial level. Among which stand out the lack of knowledge about its existence, low availability of certified seed, low rates of yield and productivity, the presence of phytopathogens due to its vegetative propagation, the lack of technology and tools to strengthen its production and the absence of focused local programs to your research and technical assistance. Therefore, a "triple helix" alliance was established between the university, private company and government in order to obtain clean traditional potato plants, characterized with ethnobotanical, agronomic, nutritional and molecular descriptors, focused on obtaining elite individuals for its agroeconomic promotion. This characterization was achieved thanks to the methodological implementation of two strategic lines, which ranged from continuous work in the laboratory and in the field through the establishment of experimental plots, to interaction with the community for the dissemination and social appropriation of knowledge. As results, effective protocols were developed to obtain clean plants of ancestral potato genotypes, using the in vitro tissue culture technique. The propagated plants were examined by microbiological techniques for the detection of fungi, bacteria and viruses. With the above, the delivery of clean native potato vitroplants to farmers in the municipalities of Ventaquemada and Chiscas in the department of Boyacá was ensured. Additionally, micropropagation ensured the molecular characterization and in vitro conservation of germplasm of native potato morphotypes of the department. In the molecular component, it was possible to establish the genetic relationships between the native potato genotypes cultivated in the department of Boyacá. This information will be the fundamental basis for the establishment of potential parental individuals in genetic improvement programs at the departmental level. In the field component, it was possible to determine the performance of each potato genotype under the conditions of two localities, finding that some of them present better development and production depending on their adaptation to the environment of the evaluated locality. It was also possible to determine the nutritional parameters of the evaluated native potato tubers. This information will allow the farmer to promote the commercialization of fresh and industrialized products, with unique organoleptic characteristics in the regional and national market. The seeding and characterization of the genotypes also creates an important milestone in the morphological identification of local genotypes. The botanical description of the flower, the arrangement of the leaves, the color of the stem, the coloration in the tuber and the plant habit are universal characters that will allow the differentiation of these plants by the community. The social appropriation of knowledge and the intervention of professionals in the different areas and disciplines, allowed the efficient use of information, giving rise to the generation of new knowledge of wide impact. Likewise, this research book shows the successful experience of linking social actors in the dissemination, generation and promotion of scientific content as a high-impact tool in favor of the promotion and encouragement of native potato genotypes. It is important to note that this book has QR codes where the reader can have direct access to the documentary entitled: "The colors and flavors of my land", as well as three explanatory and complementary capsules to the contents of the book, integrating the scientific study, the traditional knowledge of the communities, promoting the transfer of technologies for the benefit of the producers and the territory.

One hundred and f f y strains of streptomycetes were isolated from grey and dark grey forest soils, as well as from typical chernozem. T e isolated strains were analyzed in Vitro for antimicrobial activity on nutrient media and in grey forest soil against twenty-three collection pathogenic test cultures of fungi and bacteria. Four biologically active isolates with a wide spectrum of action were identif ed and deposited in the All-Russian Collection of industrial microorganisms of the National Research Centre Kurchatov Institute. T ey were identif ed and deposited under the following numbers : Streptomyces xiamenensis TB ВКПМ Ас-2204, Streptomyces anulatus TG ВКПМ Ас-2203, Streptomyces sindenensis TK ВКПМ Ас-2205, Streptomyces f avovirens TT ВКПМ АС-2202. A study of the ef ect of presowing treatment of wheat seeds by the 15-day cultural liquids of the strains S. xiamenensis TB ВКПМ Ac-2204, S. anulatus TG ВКПМ Ac-2203, S. sindenensis TK ВКПМ Ac-2205 on germination rates and infection with F. graminearum revealed that they inhibited the growth of a pathogenic fungus. Besides, they improved seed vigour and germination of wheat. T e resulting strains of soil actinomycetes can be used in biotechnology with an aim to create the new bioinoculants when dealing with phytopathogenic bacteria and fungi. T e strains can also be used for stimulating the plant growth, as well as for soil bioremediation in organic farming. T rough the application of the HPLC method, specialized antimicrobial metabolites of monosporic strain sus-pensions were identif ed. T e identif ed antibiotics are N-Butylbenzenesulfonamide, 1-(1H-Benzo[d]imidazol-2-yl)ethan-1-ol, 2-[(3S)-1-(Cyclohexylmethyl)-3-pyrrolidinyl]-1H-benzimidazole-5-carbonitrile, Cyclo(leucylprolyl), Cyclo(phenylalanyl-prolyl). T e identif ed antiseptics are Cetrimonium and Carvone. T e identif ed phytohormone is auxin indole-3-acetic acid (IAA). Observation of the dynamics of development of introduced actinobacteria under study in soil samples showed high activity of streptomycetes that use chitin. Analysis of the diversity of the prokaryotic complex of the studied soil samples with the application of the high-throughput sequencing of the conserved region of the 16S rRNA gene method revealed during the introduction of S. xiamenensis TB ВКПМ Ac-2204 that its controlling role in the microbial community is due to its antibiotic-forming activity.