Macoilin, a Conserved Nervous System–Specific ER Membrane Protein That Regulates Neuronal Excitability

Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O2 responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca2+ transients, at least in some neurons: in maco-1 mutants the O2-sensing neuron PQR is unable to generate a Ca2+ response to a rise in O2. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O2, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca2+ channels, also fails to disrupt Ca2+ responses in the PQR cell body to O2 stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca2+ channel α1 subunit, recapitulate the Ca2+ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators

Enzyme and Directed Evolution Technologies For Nerve Agent Neutralisation

Owing to the magnitude of the utilisation of organophosphorus (OPs) insecticides and the possibility of using OPs nerve agents (NA) against civilian populations, the research and development of enzymes involved in the biotransformation and detoxification of OPs has attracted considerable attention in recent years. A number of enzymes have been identified that can catalyse the hydrolysis of OPs, including nerve agents. Two of the best characterised are Pseudomonas diminuta phosphotriesterase (PTE) and PON1, a mammalian member of the Serum paraoxonase (PONs) family. These enzymes have excellent catalytic properties towards some OPs, but relatively poor activities against others. It has been possible to alter PTE substrate specificity by rational site-mutagenesis, but with little improvements on the wild-type rates. A more successful approach has been the application of directed-evolution strategies. The aim of the present work has been to create variants of PTE with an increased catalytic efficiency towards OPs nerve agents. To this end, a directed evolution platform was developed to enable screening for organophosphatase activity. This methodology relies on the screening of Escherichia coli colonies transformed with PTE-variant libraries. Twelve fluorogenic NA analogues, with a 3-chloro-7-hydroxy-4-methylcoumarin leaving group, were tested for suitability as substrates for PTEs and PON1. Included in this series were analogues of the pesticides Paraoxon and Parathion, and the chemical warfare agents DFP, Dimefox, Tabun, Sarin, Cyclosarin, Soman, VX, and Russian-VX . These chemical surrogates have a similar structure but do not share the same physico-chemical properties as the nerve agents themselves. The directed evolution platform developed and used consisted of two parts. First, partially lysed Escherichia coli colonies were screened using the fluorogenic nerve agents analogues as probes. Second, the selected (positive) clones were grown in microplates filled with liquid medium, and their organophosphatase activity was measured in vivo. Several gene libraries were synthesised in each of which four codons of the residues forming PTE’s substrate binding site were selectively randomised. The PTE variant S5a was used as template for the libraries, as it expresses at 20-fold higher level than the wild type, in bacterial hosts, while retaining its kinetic properties for the wild-type substrate, Paraoxon. These libraries were screened using analogues of Russian-VX and Parathion as probes; approximately 106 clones were screened in total. The twenty most active variants, as determined in vivo, were expressed, purified, and their kinetic parameters for Paraoxon and the NA analogues were determined. PTE-S5a itself hydrolysed 8/VX, 9/Sarin and 10/Russian-VX analogues between 2.5 and 3.5 times more readily than PTE-wt. In contrast, towards 11/Soman and 12/Cyclosarin analogues its activity, was only 70% of that of the wild type enzyme. Three of the selected clones, PTE -A (I106T), C (I106L), and H (I106T/F132V/S308A/Y309W), exhibited a higher kcat than PTE-S5a towards Paraoxon. The latter exhibited a 5-fold increased in its turnover rate (31,016 s-1); this rate is higher than that of the in vitro evolved PTE-H5 (26,294 s-1). PTE variants A (I106T), C (I106L), D (I106A/F132G), E (I106V/F132L), and F (I106L/F132lG) exhibited between 2 and 4-fold increases in their kcat/KM towards the Paraoxon analogue relative to PTE-S5a. Variants Q (G60V/I106L/ S308G), S (G60V/I106M/L303E/S308E), and T(G60V/I106S/L303P/S308G) showed between 2 and 14-fold improvements in their activities towards Russian-VX, Soman and Cyclosarin analogues. The selectivity for this latter group towards phosphonate NA analogues increased up to 107-fold, relative to the wild type PTE. Each PTE monomer binds two divalent transition metal ions via a cluster of four histidines (His-55, His-57, His-201 and His-230) and one aspartate (Asp-301). In addition, the two metal ions are linked together by a carbamate functional group, formed by the carboxylation of the e-amino group of Lys-169 and a water (or hydroxide ion) from the solvent. A case study is presented in which using both site-directed mutagenesis and directed evolution strategies, the possibility of replacing the carboxylated lysine (Lys-169) by any other residue was assessed.Gates Cambridge Trust Overseas Research Student award schem

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses

International audienceGenomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements

Patient-Patient Similarity-Based Screening of a Clinical Data Warehouse to Support Ciliopathy Diagnosis

International audienceA timely diagnosis is a key challenge for many rare diseases. As an expanding group of rare and severe monogenic disorders with a broad spectrum of clinical manifestations, ciliopathies, notably renal ciliopathies, suffer from important underdiagnosis issues. Our objective is to develop an approach for screening large-scale clinical data warehouses and detecting patients with similar clinical manifestations to those from diagnosed ciliopathy patients. We expect that the top-ranked similar patients will benefit from genetic testing for an early diagnosis. The dependence and relatedness between phenotypes were taken into account in our similarity model through medical concept embedding. The relevance of each phenotype to each patient was also considered by adjusted aggregation of phenotype similarity into patient similarity. A ranking model based on the best-subtype-average similarity was proposed to address the phenotypic overlapping and heterogeneity of ciliopathies. Our results showed that using less than one-tenth of learning sources, our language and center specific embedding provided comparable or better performances than other existing medical concept embeddings. Combined with the best-subtype-average ranking model, our patient-patient similarity-based screening approach was demonstrated effective in two large scale unbalanced datasets containing approximately 10,000 and 60,000 controls with kidney manifestations in the clinical data warehouse (about 2 and 0.4% of prevalence, respectively). Our approach will offer the opportunity to identify candidate patients who could go through genetic testing for ciliopathy. Earlier diagnosis, before irreversible end-stage kidney disease, will enable these patients to benefit from appropriate follow-up and novel treatments that could alleviate kidney dysfunction

A genotype-to-phenotype approach suggests under-reporting of single nucleotide variants in nephrocystin-1 (NPHP1) related disease (UK 100,000 Genomes Project)

Abstract Autosomal recessive whole gene deletions of nephrocystin-1 (NPHP1) result in abnormal structure and function of the primary cilia. These deletions can result in a tubulointerstitial kidney disease known as nephronophthisis and retinal (Senior–Løken syndrome) and neurological (Joubert syndrome) diseases. Nephronophthisis is a common cause of end-stage kidney disease (ESKD) in children and up to 1% of adult onset ESKD. Single nucleotide variants (SNVs) and small insertions and deletions (Indels) have been less well characterised. We used a gene pathogenicity scoring system (GenePy) and a genotype-to-phenotype approach on individuals recruited to the UK Genomics England (GEL) 100,000 Genomes Project (100kGP) (n = 78,050). This approach identified all participants with NPHP1-related diseases reported by NHS Genomics Medical Centres and an additional eight participants. Extreme NPHP1 gene scores, often underpinned by clear recessive inheritance, were observed in patients from diverse recruitment categories, including cancer, suggesting the possibility of a more widespread disease than previously appreciated. In total, ten participants had homozygous CNV deletions with eight homozygous or compound heterozygous with SNVs. Our data also reveals strong in-silico evidence that approximately 44% of NPHP1 related disease may be due to SNVs with AlphaFold structural modelling evidence for a significant impact on protein structure. This study suggests historical under-reporting of SNVS in NPHP1 related diseases compared with CNVs

Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring

International audienceStudies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

International audienceMining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility

Precios unitarios y elaboración de las propuestas técnica y económica para licitación de obra pública, con aplicación de software, en un estudio de caso.

A través de la historia el ser humano se ha preocupado por satisfacer sus necesidades básicas, además de extender sus dominios consiguiendo poder obtener una forma de lograrlo, sin lugar a dudas es el de contar con bienes materiales y económicos, los cuales están estrechamente relacionados con la moneda como forma de pago, aunque al principio de los tiempos esto se realizaba por medio de trueques de mercancías o productos, luego, con la aparición del dinero, los cambios se facilitaron ya que se pagaba realmente por el valor de un bien. De manera que el tiempo ha ido transcurriendo se han generado nuevas formas de pago desde la moneda hecha de metal, hasta la aparición de dinero electrónico. Además de que todas las áreas de estudio están influenciadas por este factor (dinero). Y cuando hablamos del dinero a cambio de alguna mercancía o producto es inevitable caer en el término “Precio” y la rama de la ingeniería civil no está exenta de esta problemática ya que el creciente desarrollo de los países ha generado mayor demanda de construcciones en cada uno de ellos. La presente investigación de tesis, corresponde a: “Precios unitarios y elaboración de las propuestas técnica y económica para licitación de obra pública”. Es indispensable para un Ingeniero Civil comprender que La licitación Públicaes un procedimiento administrativo de preparación de la voluntad contractual, por el que un ente público en ejercicio de la función administrativa invita a los interesados para que, sujetándose al pliego de condiciones (Bases de la licitación), formulen propuestas (Técnica y Económica) de entre las cuales seleccionará la más conveniente (Adjudicación). Cuando es necesario contratar obra pública, existen leyes que obligan a los entes gubernamentales a seguir un proceso legalmente definido por el derecho administrativo. En México, el Artículo 134 Constitucional determina como el gobierno debe realizar las adquisiciones y contratación de obra pública; de esa Ley se deriva la Ley de Adquisiciones, Arrendamiento y Servicios del Sector Público y Ley de Obras Públicas y Servicios relacionados con las mismas