Disseny d'un sistema de filtrat d'àudio

Tant el medi transmissor com els equips d'enregistrament o reproducció de so introdueixen components de soroll d'alta freqüència als senyals. En aquest treball de final de carrera (TFC), s'ha dissenyat i implementat un sistema de filtrat d'àudio encaminat a filtrar aquestes components d'alta freqüència. Donat que l'oïda humana no pot percebre sons de més de 20 kHz, s'ha considerat aquest límit com a freqüència màxima a mantenir en la senyal. S'ha començat estudiant el senyal problema a través del seu espectre de freqüències simulat mitjançant la transformada discreta de Fourier (DFT, en anglès). Una vegada identificades les components d'alta freqüència a atenuar, s'han estudiat les diferents opcions de filtre passabaix. Inicialment, s'ha valorat la possibilitat del disseny de filtres analògics de Butterworth o Chebyshev, o de filtres digitals de tipus IIR (Infinite Impulse Response) basats en els primers. Tanmateix, malgrat assolir les especificacions en magnitud, mitjançant aquest filtres no s'obté una fase lineal en la banda de pas. Per això, s'ha realitzat un disseny de filtre digital tipus FIR (Finite Infinite Response) que compleix estrictament amb les especificacions i presenta una fase lineal en la banda de pas. S'ha simulat el comportament d'aquest filtre amb el senyal problema per tal d'assegurar el seu correcte funcionament. A continuació, s'ha implementat aquest últim disseny en llenguatge C i compilat per un microcontrolador de l'empresa Microchip. S'han realitzat proves de simulació mitjançant Stimulus del programa MPLAB. En definitiva, s'ha dissenyat un filtre passabaix de tipus FIR per acondicionar una senyal d'àudio que posteriorment s'ha implementat en un microcontrolador de Microchip.Tanto el medio transmisor como los equipos de grabación o reproducción de sonido introducen componentes de ruido de alta frecuencia en las señales. En este trabajo de final de carrera (TFC), ha diseñado e implementado un sistema de filtrado de audio encaminado a filtrar estos componentes de alta frecuencia. Dado que el oído humano no puede percibir sonidos de más de 20 kHz, se ha considerado este límite como frecuencia máxima a mantener en la señal.Transmitting medium and recording and sound reproduction equipments put components of high frequency noise into the signals. In this bachelor thesis, it has been designed and implemented an audio filtering system to filter these high frequency components. Since the human ear can not hear sounds above 20 kHz, this limit was considered as maximum frequency to keep the signal

Big complexity in a minimal bacterium

With only 689 genes Mycoplasma pneumoniae (M. pneumoniae) is among the simplest known organisms. Because of this simplicity, mycoplasma represents an attractive organism for systems-wide analyses. Such approaches aiming at the whole quantitative understanding of an entire organism are expected to illustrate the basic principles of life. Strand-specific tiling arrays complemented by transcriptome sequencing, were combined with more than 252 spotted arrays to study M. pneumoniae transcriptional organization. An important presence of alternative transcripts (42%) within operons and a high frequency of antisense RNA (89) were detected. Metabolism was also studied in detail. A manually curated metabolic network allowed the definition of a minimal medium with 19 essential nutrients. This has been complemented with measurements of biomass indicators, metabolites and fluxes. Integration with transcriptional profiling has provided keys in the metabolic regulation. Protein organization and interactions have been addressed systematically by Tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The biochemical analysis revealed 178 protein complexes which have been complemented by structural models, single-article electron microscopy and electron tomography. By integrating the datasets from these different approaches, we show that this small bacterium harbors an unexpected complexity with features such as the frequent occurrence of alternative transcripts and antisense RNA, a small but tightly controlled metabolic network and a high level of proteome organization.Amb només 689 gens Mycoplasma pneumoniae es troba entre els organismes més simples que es coneixen. Degut a aquesta simplicitat, mycoplasma representa un organisme atractiu per dur a terme estudis a nivell genòmic. S'espera d'aquests treballs que pretenen descriure de manera quantitativa l'organisme sencer que ajudin a entendre els principis bàsics de la vida. Per tal l'estudiar amb profunditat del transcriptoma, s'ha fet ús d'una combinació de dades de "tiling arrays" amb especificitat de cadena, ultraseqüenciació i més de 252 microarrays. Després d'analitzar els resultats s'ha detectat una alta presència de transcrits alternatius (42%) dintre operons i una alt contingut de ARN de tipus "antisense" (89). També s'ha realitzat un estudi detallat del metabolisme. S'ha revisat i completat manualment el mapa metabòlic de M. pneumoniae, fet que ha permès el disseny d'un medi mínim amb l'ús de 19 ingredients essencials. El mapa s'ha completat amb diferents mesures d'indicadors de biomassa, metabòlits i fluxos. També s'ha estudiat la regulació de metabolisme mitjançant microarrays. Per altra banda, s'han mesurat sistemàticament les interaccions proteïna-proteïna mitjançant "Tandem affinity purification-mass spectrometry (TAP-MS)". Aquest anàlisis ha detectat 178 complexes diferents, els quals han estat complementats amb models estructurals, microscòpia electrònica i tomografia electrònica. Mitjançant la integració d'aquestes col·leccions de dades, es pot mostrar que aquest petit bacteri amaga un inesperada complexitat amb característiques com la freqüència de transcrits alternatius i ARN "antisense", una xarxa metabòlica petita però fortament controlada i una alta organització del proteoma

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494. We thank H. Stutzman for assistance with cloning plasmids, and Z. Herbert and M. Berkeley from the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute for assistance with NextSeq sequencing

Heterologous erythromycin production across strain and plasmid construction

The establishment of erythromycin production within the heterologous host E. coli marked an accomplishment in genetic transfer capacity. Namely, over 20 genes and 50 kb of DNA was introduced to E. coli for successful heterologous biosynthetic reconstitution. However, the prospect for production levels that approach those of the native host requires the application of engineering tools associated with E. coli. In this report, metabolic and genomic engineering were implemented to improve the E. coli cellular background and the plasmid platform supporting heterologous erythromycin formation. Results include improved plasmid stability and metabolic support for biosynthetic product formation. Specifically, the new plasmid design for erythromycin formation allowed for ≥89% stability relative to current standards (20% stability). In addition, the new strain (termed LF01) designed to improve carbon flow to the erythromycin biosynthetic pathway provided a 400% improvement in titer level. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:271-276, 2018

DNA damage protection for enhanced bacterial survival under simulated low earth orbit environmental conditions in Escherichia coli

Some organisms have shown the ability to naturally survive in extreme environments, even outer space. Some of these have natural mechanisms to resist severe DNA damage from conditions such as ionizing and non-ionizing radiation, extreme temperatures, and low pressures or vacuum. A good example can be found in Deinococcus radiodurans, which was exposed to severe conditions such as those listed in the Exposure Facility of the International Space Station (ISS) for up to three years. Another example are tardigrades (Ramazzottius varieornatus) which are some of the most resilient animals known. In this study, the survival under simulated Low earth Orbit (LEO) environmental conditions was tested in Escherichia coli. The radiation resistance of this bacteria was enhanced using the Dsup gene from R. varieornatus, and two more genes from D. radiodurans involved in DNA damage repair, RecA and uvrD. The enhanced survival to wide ranges of temperatures and low pressures was then tested in the new strains. This research constitutes a first step in the creation of new bacterial strains engineered to survive severe conditions and adapting existing species for their survival in remote environments, including extra-terrestrial habitats. These strains could be key for the development of environments hospitable to life and could be of use for ecological restoration and space exploration. In addition, studying the efficacy and the functioning of the DNA repair mechanisms used in this study could be beneficial for medical and life sciences engineering

Establishing a cell-free transcription-translation platform for Cutibacterium acnes to prototype engineered metabolic and synthetic biology

Data de publicació electrònica: 31-12-2021In the past few years, new bacterial-cell-free transcription-translation systems have emerged as potent and quick platforms for protein production as well as for prototyping of DNA regulatory elements, genetic circuits, and metabolic pathways. The Gram-positive commensal Cutibacterium acnes is one of the most abundant bacteria present in the human skin microbiome. However, it has recently been reported that some C. acnes phylotypes can be associated with common inflammatory skin conditions, such as acne vulgaris, whereas others seem to play a protective role, acting as possible "skin probiotics". This fact has made C. acnes become a bacterial model of interest for the cosmetic industry. In the present study we report for the first time the development and optimization of a C. acnes-based cell-free system (CFS) that is able to produce 85 μg/mL firefly luciferase. We highlight the importance of harvesting the bacterial pellet in mid log phase and maintaining CFS reactions at 30 °C and physiological pH to obtain the optimal yield. Additionally, a C. acnes promoter library was engineered to compare coupled in vitro TX-TL activities, and a temperature biosensor was tested, demonstrating the wide range of applications of this toolkit in the synthetic biology field.This work was funded by the Office of Naval Research (Award N62909-18-1-2155). M.-J.F was funded by a Juan de la Cierva Fellowship (Spanish Government). N.K. was funded by a Maria Maetzu-UPF Fellowship (Catalan Government). G.N. was funded by an FI Fellowship (AGAUR-Catalan Govern- ment). This project received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie Grant Agreement 882387 (to J.S.-M.)

From dysbiosis to healthy skin: major contributions of Cutibacterium acnes to skin homeostasis

Cutibacterium acnes is the most abundant bacterium living in human, healthy and sebum-rich skin sites, such as the face and the back. This bacterium is adapted to this specific environment and therefore could have a major role in local skin homeostasis. To assess the role of this bacterium in healthy skin, this review focused on (i) the abundance of C. acnes in the skin microbiome of healthy skin and skin disorders, (ii) its major contributions to human skin health, and (iii) skin commensals used as probiotics to alleviate skin disorders. The loss of C. acnes relative abundance and/or clonal diversity is frequently associated with skin disorders such as acne, atopic dermatitis, rosacea, and psoriasis. C. acnes, and the diversity of its clonal population, contributes actively to the normal biophysiological skin functions through, for example, lipid modulation, niche competition and oxidative stress mitigation. Compared to gut probiotics, limited dermatological studies have investigated skin probiotics with skin commensal strains, highlighting their unexplored potential

INSERT-seq enables high-resolution mapping of genomically integrated DNA using Nanopore sequencing

Comprehensive characterisation of genome engineering technologies is relevant for their development and safe use in human gene therapy. Short-read based methods can overlook insertion events in repetitive regions. We develop INSERT-seq, a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration. The experimental pipeline improves the number of mappable insertions at repetitive regions by 4.8-7.3% and larger repeats are processed with a computational peak calling pipeline. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex vivo and in vivo samples.We thank funding received from UPGRADE (European Union Horizon 2020, grant agreement No 825825), Fundación Ramón Areces (“Advanced gene editing technologies to restore LAMA2 on merosin-deficient congenital muscular dystrophy type 1A”), MdM projecte de recerca “Unidad de Excelencia María de Maeztu”, funded by the AEI (CEX2018-000792-M)