CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single-molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations

Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

Prebiotic effects of olive pomace powders in the gut: In vitro evaluation of the inhibition of adhesion of pathogens, prebiotic and antioxidant effects

Supplementary data related to this article can be found at https:// doi.org/10.1016/j.foodhyd.2020.106312Olive pomace is a biowaste rich in polyphenols and insoluble dietary fibre with high potential to develop new value chains towards a sustainable and circular bioeconomy. Regarding gut health, olive pomace phenolics and insoluble dietary fibre (after possible fermentation) could act as antioxidants, antimicrobial and prebiotic agents. These potential beneficial effects on the gut were analysed for two powders from olive pomace: liquid-enriched powder (LOPP) - mostly source of phenolics - and pulp-enriched powder (POPP) - main source of insoluble dietary fibre. LOPP and POPP were subjected to an in vitro simulated gastrointestinal digestion followed by in vitro faecal fermentation. The undigested fraction retained in the colon was analysed regarding its potential antioxidant, antimicrobial and prebiotic effects. LOPP and POPP did not impact the gut microbiota diversity negatively, showing a similar ratio of Firmicutes/Bacteroidetes compared to a positive control (FOS). LOPP exhibit a positive (similar to FOS) effect on the Prevotella spp./Bacteroides spp. ratio. Both powders also promoted more the production of short-chain fatty acids (mainly acetate?>?butyrate?>?propionate) than FOS. Both powders showed also significant total phenolic content and oxygen radical absorbance capacity during faecal fermentation until 48?h. Besides that, these powders showed mucin-adhesion inhibition ability against pathogens, principally POPP against Bacillus cereus (22.03?±?2.45%) and Listeria monocytogenes (20.01?±?1.93%). This study demonstrates that olive pomace powders have prebiotic effects on microbiota, including the stimulation of short-chain fatty acids production, potential antioxidant and antimicrobial activity which could improve human gut health.Tˆania I. B. Ribeiro thanks the FCT - Fundação para a Ciência e Tecnologia, Portugal and Association BLC3 – Technology and Innovation Campus, Centre Bio R&D Unit for the PhD Grant SFRH/BDE/108271/2015. This work was supported by National Funds from FCT - Fundação para a Ciência e a Tecnologia, Portugal through the project MULTI-BIOREFINERY - SAICTPAC/0040/2015 (POCI-01-0145-FEDER- 016403). We would also like to thank the scientific collaboration under the FCT project UID/Multi/50016/2019.info:eu-repo/semantics/publishedVersio

Long Noncoding RNA NIHCOLE Promotes Ligation Efficiency of DNA Double-Strand Breaks in Hepatocellular Carcinoma

[Abstract] Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability.[Significance] This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.This work was supported by the European FEDER funding (to the activities of the groups directed by P. Fortes, O. Llorca, and F. Moreno-Herrero) and grants from the Ministry of Economy and Competitiveness [SAF2015-70971-R to P. Fortes and BFU2017-83794-P (AEI/FEDER, UE) to F. Moreno-Herrero)]; MCIU/AEI/FEDER/UE (RTI2018-101759-B-I00 to P. Fortes), NIH program (CA92584 to S.P. Lees-Miller), Ligue National Contre le Cancer, Équipe Labellisée and ITMO Cancer: Consortium HETCOLI (to J. Zucman-Rossi), NIH program (P01CA092584 to G. Williams), NSERC (RGPIN-2018-04327 to G. Williams), and CFI (RCP-18-023-SEG to G. Williams), Gobierno de Navarra (33/2015 to P. Fortes), Scientific Foundation of the Spanish Association Against Cancer (AECC IDEAS20169FORT to P. Fortes); Fondo de Investigación Sanitaria (PI19/00742 to B. Sangro), financed by the National Institute of Health Carlos III and FEDER. CNIO and CIBERehd are funded by the National Institute of Health Carlos III. J.P. Unfried was a recipient of a University of Navarra's Asociación de Amigos fellowship. L. Prats-Mari is a recipient of a PFIS fellowship (FI20/00074) by the National Institute of Health Carlos III and FSE "Investing in Your Future." This work was also funded by grants from the Autonomous Region of Madrid (Tec4Bio—S2018/NMT-4443 and NanoBioCancer—Y2018/BIO-4747 to O. Llorca and F. Moreno-Herrero) and co-funded by the European Social Fund. F. Moreno-Herrero acknowledges support from the European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (grant agreement 681299). The GTEx Project was supported by the NIH and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.Peer reviewe

Magnetic Tweezers Data - Lokanathan Balaji et al. "DNA binding and bridging by human CtIP in the healthy and diseased states"

<p>These timecourses and datasets correspond to the publication: Lokanathan-Balaji et al. “DNA binding and bridging by human CtIP in the healthy and diseased states” bioRxiv 2023 (https://doi.org/10.1101/2023.12.14.571649)<br>----<br><span><a href="../api/records/10499058/draft/files/Fig4+5_Datasets.zip/content" target="_blank" rel="noopener noreferrer">Fig4+5_Datasets.zip</a></span> and <span><a href="../api/records/10499058/draft/files/Fig4+5_Timecourses.zip/content" target="_blank" rel="noopener noreferrer">Fig4+5_Timecourses.zip</a></span> folders contain all the datasets and the individual timecourses, respectively, for all the conditions addressed in Figures 4 and 5:<br>    - Bare DNA<br>    - DNA + wild-type CtIP <br>    - DNA + lambda CtIP <br>    - DNA + R100W CtIP <br>    - DNA + deltaC608 CtIP <br>    - DNA + deltaC782 CtIP <br>    - DNA + deltaN500 CtIP <br>    - DNA + deltaN790 CtIP </p> <p><span><a href="../api/records/10499058/draft/files/Fig4+5_Datasets.zip/content" target="_blank" rel="noopener noreferrer">Supp_FigS4_Datasets.zip</a> and <a href="../api/records/10499058/draft/files/Supp_FigS4_Timecourses.zip/content" target="_blank" rel="noopener noreferrer">Supp_FigS4_Timecourses.zip</a></span> folders contain all the datasets and the individual timecourses, respectively, for all the conditions addressed in Supplementary Figure S4:<br>    - fork-end branched DNA (forked) + wild-type CtIP<br>    - fork-end branched DNA (forked) + lambda CtIP</p&gt

Contribuição para o estudo das pastagens sob coberto de folhosas

Relatório de atividade do aluno estagiário do curso de Engenheiro Silvicultor - Instituto Superior de AgronomiaN/

Aplicação de Standard Work e de outras ferramentas de Lean Production numa empresa de elevadores

Dissertação de mestrado integrado em Engenharia e Gestão IndustrialO presente trabalho enquadra-se no âmbito do Projeto de Dissertação inserido no Mestrado Integrado em Engenharia e Gestão Industrial da Universidade do Minho, 5º ano, 2º semestre. Este projeto teve como principal objetivo a implementação de Standard Work e outras ferramentas do modelo organizacional de produção Lean Production numa secção de montagem de portas da empresa de produção de elevadores Schmitt + Sohn Elevadores. A metodologia de investigação usada foi a Action Research. Esta investigação iniciou-se com uma revisão bibliográfica sobre Lean Production, história, princípios, benefícios da implementação e principais ferramentas usadas neste modelo organizacional, como os 5S, a Gestão Visual, o Kaizen ou o Standard Work. O diagnóstico da empresa foi iniciado realizando uma apresentação e caracterização da empresa mais abrangente seguida por uma descrição e análise mais detalhada do funcionamento da seção objeto de estudo deste projeto. Tendo em consideração o elevado número de produtos produzidos foi necessário escolher apenas um para fazer uma análise mais minuciosa. Deste modo, analisaram-se vários parâmetros (cadeia de valor, atividades nos postos de trabalho, tempos, não conformidades, competências, entre outros) e identificaram-se aspetos que poderiam ser melhorados. A partir do reconhecimento dos problemas encontrados identificaram-se algumas propostas que poderiam soluciona-los, tendo algumas sido implementadas e outras não. Tendo por base a ideologia do Lean “doing more with less” a grande maioria das propostas apresentadas não trouxe custos significativos para a empresa conseguindo-se melhorias interessantes. Neste sentido, muitas das propostas apresentadas estão relacionadas com a melhor organização do espaço fabril e com a criação de documentos de modo a criar um ambiente mais harmonioso, saudável e seguro para todos. As várias propostas de melhoria sugeridas tiveram impactos positivos diversos como eliminar várias distâncias percorridas pelos operadores (90m), diminuição do tempo despendido na procura de materiais (80%), diminuição do idle time (92%) e aumento da capacidade do supermercado (50%).This project was developed in the framework of the Dissertation Project within in the Master in Industrial Engineering and Management of the University of Minho, 5th year, 2nd semester. This project had the main objective of implementation of Standard Work and other tools and techniques of the organizational production model Lean Production in the door assembly section of the elevators company Schmitt + Sohn Elevadores. The research methodology used was the Action Research. This research began with a bibliographic review of Lean Production, history, principles, implementation and benefits, the main tools used in this organizational model, such as 5S, Visual Management, Kaizen and Standard Work. The diagnosis of the company started by making a presentation and characterization of the company followed by a more comprehensive description and detailed analysis of the functioning of the section object of this project. Given the large number of products produced it was necessary to choose only one to do a more thorough analysis. Thus, were analyzed several parameters (value stream, activities in the workplace, time, non-compliance, competencies, etc.) to identify the aspects that could be improved. Based on the recognition of the problems found were identified some proposals that could solve them, some have been implemented and others have not. Based on the Lean ideology of "doing more with less" the vast majority of the proposals did not bring significant costs to the company managing to have interesting improvements. In this sense, many of the proposals are related to the better organization of factory space and the creation of documents in order to create a more harmonious, healthy and safe for everyone workplace. The various proposals for improvement suggested brought many positive impacts such as eliminating various distances traveled by the operators (90m), reducing time spent searching for materials (80%), reduced idle time (92%) and increasing the capacity of the supermarket (50%)

Study on the influence of common working postures on individuals’ comfort and anthropometric data

Tese de Doutoramento - Programa Doutoral em Engenharia Industrial e de SistemasWork occupies a large portion of peoples’ lives. The intrinsic factors associated with work, such as the difficulty of the tasks to perform, can themselves cause much stress to the workers. When the extrinsic factors, such as the environment or the working conditions, are also inadequate, working becomes even more difficult. These negative issues may limit workers’ performance and put their comfort and health at risk, compromising the quality of their task, performance and productivity. Therefore, considering ergonomics interventions in every job is crucial. Issues where interventions are most certainly needed are the working postures, namely, by studying how they affect the human body and how they limit the work activities. As such, the main purpose of the research carried out in this thesis was to assess the influence that both sitting and standing working postures have on the changes of workers’ anthropometric data, as well as to assess their perceived comfort. The path taken to accomplish this goal required additional smaller objectives to be accomplished as well. First of all, it was necessary to select the best way to collect the necessary data. To do this, two anthropometric data collection techniques were compared – the traditional manual anthropometry and the 3D anthropometry. When put side by side, it was possible to see a significant difference between the two data acquisition methods for almost all body measurements. However, it was concluded that these differences could be caused by many factors, such as the involuntary body sway or the pressure exerted by the equipment, and not necessarily mean that the methods are inadequate. Indeed, the precision and reliability tests showed that both methods are good. Nevertheless, the 3D data, obtained with the specific body scanner used in this study, should only be used for applications that are not very demanding. Then, came the analysis of the body modifications that occur with each working posture. A pilot study was performed that tried to identify the changes that happen in the human body during a day of work (e.g. leg swelling and decrease of stature, much reported in the literature). From the obtained data, it was possible to verify that the modifications really do happen, but at a very small scale. After this, the study evolved to the quantification of the instant modifications of the body when common dynamic postures are assumed. At this point, the results obtained prompted a new focus on the discomfort felt by workers caused by the improper work-wear. A questionnaire filled by active workers with different types of work requirements demonstrated that there are many postures that are limited because of the clothes worn. It is the lower part of the garment that they felt more uncomfortable with but it is the upper part that is more restrictive. This helped to make clear the need to act and to create a product that can make people feel less restricted and more comfortable. As such, two prototypes of a shirt with simple alterations were made, one had the application of an elastic mesh in the armhole area and the other had the application of the same mesh on the back area. The tests performed to validate these prototypes showed that, when compared to a regular model designed for the static standing posture, the compression forces decreases and the stretch ability increases, meaning that they provide more flexibility and fewer limitations of movements. With all the objectives accomplished it can be said that the results and findings obtained with this thesis can bring many contributions to the scientific community, alongside with important practical information that can be used worldwide by companies or individual people.O trabalho ocupa uma grande parte da vida das pessoas. Os fatores intrínsecos associados ao trabalho, como a dificuldade das tarefas a executar, podem por si só causar muito stresse aos trabalhadores. Quando os fatores extrínsecos, como o ambiente ou as condições de trabalho, são também inadequados o trabalho torna-se ainda mais difícil. Estes aspetos negativos podem limitar a capacidade de trabalho dos trabalhadores e colocar em risco o seu conforto e saúde, comprometendo a qualidade das suas tarefas e a sua produtividade. Como tal, é crucial ter em conta os aspetos ergonómicos associados aos locais e características do trabalho. Há alguns aspetos onde intervenções são necessárias, como é o caso das posturas de trabalho, em particular tentando compreender como é que estas afetam o corpo humano e como é que limitam as atividades do trabalho. Assim, o objetivo desta tese foi avaliar a influência que as posturas de trabalho, de pé e sentado, têm nas modificações dos dados antropométricos dos trabalhadores e também avaliar a sua perceção de conforto. O caminho seguido para atingir este objetivo exigiu que outros objetivos fossem também alcançados. Em primeiro lugar foi necessário selecionar a melhor forma de recolher os dados necessários. Para tal, foram comparados dois métodos de recolha de dados antropométricos – a recolha manual de dados antropométricos tradicional e de dados antropométricos 3D. Quando se compararam os resultados obtidos foi possível observar que existem diferenças significativas entre os dados adquiridos com os diferentes métodos para quase todas as medidas do corpo. No entanto, concluiu-se que estas diferenças podem dever-se a muitos fatores, como o balanço involuntário do corpo humano ou a pressão exercida pelos equipamentos de medição, e não necessariamente aos métodos em si. De facto, os testes de precisão e fiabilidade mostraram que ambos os métodos são bons. Porém, os dados 3D, obtidos com o body scanner específico usado para este estudo, só devem ser usados para aplicações pouco exigentes. Na fase seguinte foi feita a análise das modificações que ocorrem no corpo com cada postura de trabalho. Foi feito um teste piloto para tentar identificar as alterações que se dão no corpo humano durante um dia de trabalho (e.g. inchaço das pernas e a diminuição da estatura, muito reportados na literatura). Com os dados obtidos, foi ainda possível verificar que as modificações ocorrem de facto, mas a uma escala muito pequena. Depois disto, o estudo evoluiu para a quantificação das modificações instantâneas no corpo quando são adotadas posturas dinâmicas. Nesta fase do estudo, os resultados obtidos fizeram as atenções voltar-se para o desconforto causado pelo vestuário de trabalho desapropriado. Um questionário preenchido por trabalhadores com diferentes tipos de atividades demonstrou que há muitas posturas que são limitadas pelo vestuário usado. É com a parte inferior do vestuário que se sentem mais desconfortáveis mas é na parte superior que sentem mais restrições de movimentos. Isto ajudou a clarificar a necessidade de atuar e criar um produto que permite às pessoas sentirem-se menos restringidas e mais confortáveis. Assim, dois protótipos de uma camisa com pequenas alterações foram criados, um com uma malha elástica na cava e outro com a mesma malha nas costas. Os testes para validar estes protótipos mostraram que, quando comparados com um modelo normal desenhado com medidas estáticas, as forças de compressão diminuem e a extensibilidade aumenta, comprovando maior flexibilidade e menor limitação dos movimentos. Com todos os objetivos cumpridos pode-se dizer que os resultados desta tese podem trazer bastantes contributos para a comunidade científica, juntamente com informações práticas que podem ser usadas mundialmente por empresas ou pessoas individuais

Recent insights into eukaryotic double-strand DNA break repair unveiled by single-molecule methods

Genome integrity and maintenance are essential for the viability of all organisms. A wide variety of DNA damage types have been described, but double-strand breaks (DSBs) stand out as one of the most toxic DNA lesions. Two major pathways account for the repair of DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Both pathways involve complex DNA transactions catalyzed by proteins that sequentially or cooperatively work to repair the damage. Single-molecule methods allow visualization of these complex transactions and characterization of the protein:DNA intermediates of DNA repair, ultimately allowing a comprehensive breakdown of the mechanisms underlying each pathway. We review current understanding of the HR and NHEJ responses to DSBs in eukaryotic cells, with a particular emphasis on recent advances through the use of single-molecule techniques.Work in the laboratory of F.M-H. was supported by grants PID2020-112998GB-I00 from the Ministerio de Ciencia e Innovación (MICINN)/Agencia Estatal de Investigación (AEI/10.13039/501100011033)_FEDER, EU, cofunded by the European Regional Development Fund (ERDF); grants Y2018/BIO4747 and P2018/NMT4443 from the Autonomous Region of Madrid and cofunded by the European Social Fund (ESF) and the ERDF; and grant EUREXCEL 951214 funded by CSIC. Work in the laboratory of M.S.D. was supported by the Wellcome Trust (100401/Z/12/Z).Peer reviewe