Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

<p>Abstract</p> <p>Background</p> <p>The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients.</p> <p>Methods</p> <p>We developed a novel <it>in vitro </it>T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay) as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients.</p> <p>Results</p> <p>In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100_154–162reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~10¹⁰cells).</p> <p>Conclusion</p> <p>This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.</p

Role of vegetated coastal ecosystems as nitrogen and phosphorous filters and sinks in the coasts of Saudi Arabia

Vegetated coastal ecosystems along the Red Sea and Arabian Gulf coasts of Saudi Arabia thrive in an extremely arid and oligotrophic environment, with high seawater temperatures and salinity. Mangrove, seagrass and saltmarsh ecosystems have been shown to act as efficient sinks of sediment organic carbon, earning these vegetated ecosystems the moniker \u27blue carbon\u27 ecosystems. However, their role as nitrogen and phosphorus (N and P) sinks remains poorly understood. In this study, we examine the capacity of blue carbon ecosystems to trap and store nitrogen and phosphorous in their sediments in the central Red Sea and Arabian Gulf. We estimated the N and P stocks (in 0.2 m thick-sediments) and accumulation rates (for the last century based on 210Pb and for the last millennia based on 14C) in mangrove, seagrass and saltmarsh sediments from eight locations along the coast of Saudi Arabia (81 cores in total). The N and P stocks contained in the top 20 cm sediments ranged from 61 g N m-2 in Red Sea seagrass to 265 g N m-2 in the Gulf saltmarshes and from 70 g P m-2 in Red Sea seagrass meadows and mangroves to 58 g P m-2 in the Gulf saltmarshes. The short-term N and P accumulation rates ranged from 0.09 mg N cm-2 yr-1 in Red Sea seagrass to 0.38 mg N cm-2 yr-1 in Gulf mangrove, and from 0.027 mg P cm-2 yr-1 in the Gulf seagrass to 0.092 mg P cm-2 yr-1 in Red Sea mangroves. Short-term N and P accumulation rates were up to 10-fold higher than long-term accumulation rates, highlighting increasing sequestration of N and P over the past century, likely due to anthropogenic activities such as coastal development and wastewater inputs. © 2020 The Author(s). Published by IOP Publishing Ltd

Simulation of the evolution of floor covering ceramic tiles during the firing

In the context of the firing of ceramic tiles the problem of simulating the final shape of the body is relevant because several defects can occur and the tile can be rejected if the conditions of the firing are inadequate for the geometry and materials of the tile -- The existing literature on this problem indicates that previous works present limitations in aspects such as not using a model characteristic of ceramics at high temperatures and oversimplifying the problem -- As a response to such shortcomings, this article presents a simulation with a 3-dimensional Norton’s model, which overcomes the difficulties because it is characteristic of ceramics at high temperatures -- The results of our simulated experiments show advantages with respect to the identification of the mechanisms that contribute to the final shape of the body -- Our work is able to divide the history of temperatures in stages where the evolution of the thermal, elastic and creep deformations is simplified and meaningful -- That is achieved because our work found that curvature is the most descriptive parameter of the simulation, the most important contribution of this article -- Future work is to be realized in the creation of a model that takes into account that the shrinkage is dependent on the history of temperatures -- The main shortcoming of the paper is the lack of physical experiments to corroborate the simulation

Wildfires enhance phytoplankton production in tropical oceans

Unidad de excelencia María de Maeztu CEX2019-000940-MWildfire magnitude and frequency have greatly escalated on a global scale. Wildfire products rich in biogenic elements can enter the ocean through atmospheric and river inputs, but their contribution to marine phytoplankton production is poorly understood. Here, using geochemical paleo-reconstructions, a century-long relationship between wildfire magnitude and marine phytoplankton production is established in a fire-prone region of Kimberley coast, Australia. A positive correlation is identified between wildfire and phytoplankton production on a decadal scale. The importance of wildfire on marine phytoplankton production is statistically higher than that of tropical cyclones and rainfall, when strong El Niño Southern Oscillation coincides with the positive phase of Indian Ocean Dipole. Interdecadal chlorophyll-a variation along the Kimberley coast validates the spatial connection of this phenomenon. Findings from this study suggest that the role of additional nutrients from wildfires has to be considered when projecting impacts of global warming on marine phytoplankton production

Wildfires enhance phytoplankton production in tropical oceans

Wildfire magnitude and frequency have greatly escalated on a global scale. Wildfire products rich in biogenic elements can enter the ocean through atmospheric and river inputs, but their contribution to marine phytoplankton production is poorly understood. Here, using geochemical paleo-reconstructions, a century-long relationship between wildfire magnitude and marine phytoplankton production is established in a fire-prone region of Kimberley coast, Australia. A positive correlation is identified between wildfire and phytoplankton production on a decadal scale. The importance of wildfire on marine phytoplankton production is statistically higher than that of tropical cyclones and rainfall, when strong El Niño Southern Oscillation coincides with the positive phase of Indian Ocean Dipole. Interdecadal chlorophyll-a variation along the Kimberley coast validates the spatial connection of this phenomenon. Findings from this study suggest that the role of additional nutrients from wildfires has to be considered when projecting impacts of global warming on marine phytoplankton production

Gut-Brain Axis: Role of Microbiota in Parkinson’s Disease and Multiple Sclerosis

It has recently been discovered that the digestive tract is lined with about 100 million nerve cells; the digestive tract has been baptized, metaphorically speaking, as “the second brain,” which contains a multitude of neurotransmitters, viruses, and bacteria that help regulate our emotional state. This second brain, known as the enteric nervous system, is a unique anatomical unit that extends from the esophagus to the anus. Like the nervous system, it produces a whole series of psychoactive substances, such as serotonin, dopamine, and opioids for pain, and synthesizes benzodiazepines. In it, we find the microbiota: a set of microorganisms (viruses and bacteria). Together with the brain, the microbiota directly influences mood, character, or sleep. Knowledge about the possible relationship of the microbiota with frequent neurological diseases is still just beginning. Recently, possible changes in the microbiota have been linked to the onset of Parkinson’s disease (PD). Also, today, we know that there are differences between the microbiota of healthy people and people with multiple sclerosis and that these differences have also been related to the disease and its evolution

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Alimentação popular em São Paulo (1920 a 1950): políticas públicas, discursos técnicos e práticas profissionais

This article discusses how the concept of lower-class eating habits came about and developed in the intellectual circles of São Paulo during the first half of the 20th century. It starts by reconstructing the elements of the debate around the income and ignorance of the underprivileged as the main reasons behind their bad eating habits. Then, it looks at the focal points for interventions and public policies proposed by the government to deal with the problem thus identified, namely: training methods to produce sanitation counselors capable of offering dietary guidance as well; popular educational campaigns and new learning sites in addition to schools (e.g. healthcare centers and households); lunch and other means of offering food at schools; and diagnostic studies about food intake and eating habits among laborers. Because they were translated into technical and scientific language, the proposals and policies implemented in São Paulo left traces in a variety of supporting documents and media (photographs, primers, posters, inquiry notebooks, and academic literature).O artigo discute a construção da idéia de alimentação popular nos meios intelectuais em São Paulo, na primeira metade do século XX. Para isso, reconstitui, como motivos da má alimentação, elementos do debate em torno da renda e da ignorância dos mais pobres. Identificado o problema, as propostas de intervenção e as políticas públicas concentraram-se em alguns setores, abordados neste trabalho: métodos para a formação de educadores sanitários aptos a atuar também na educação alimentar; campanhas de instrução popular e criação de novos lugares de aprendizado (além das escolas, os centros de saúde e os lares); merenda escolar e outras alternativas de alimentação nas escolas; e diagnósticos referentes ao conteúdo e à forma da alimentação dos operários. Traduzidas em discurso técnico-científicos, as propostas e políticas implementadas na cidade deixaram indícios em documentação de suporte e tipologia variados (fotografias, cartilhas, cartazes, cadernetas de inquéritos e textos acadêmicos).Universidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

The Response of Lactococcus lactis to Membrane Protein Production

Background: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. Methodology and Findings: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. Conclusions: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.