Revealing microbial recognition by specific antibodies

Background: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system. Results: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin. Conclusions: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

[Background] Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva.[Results] We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously.[Conclusions] Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.The work (proposal: 10.46936/10.25585/60000940) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. RS, MB, JL, and TW are supported by NIH grant R01AI168993. The John Templeton Foundation (grant nos. 51250 and 60973) supported TT and SVD, and the Gordon and Betty Moore Foundation grants (GBMF7617 and GBMF9340) supported SVD. MD is supported by the Generalitat Valenciana program GenT grant number CDEIGENT/2021/008. SPE is supported by a FPU grant from the Spanish Ministry of Universities (Reference: FPU20/05756).Peer reviewe

Defining the human gut host-phage network through single-cell viral tagging.

Viral discovery is accelerating at an unprecedented rate due to continuing advances in culture-independent sequence-based analyses. One important facet of this discovery is identification of the hosts of these recently characterized uncultured viruses. To this end, we have adapted the viral tagging approach, which bypasses the need for culture-based methods to identify host-phage pairings. Fluorescently labelled anonymous virions adsorb to unlabelled anonymous bacterial host cells, which are then individually sorted as host-phage pairs, followed by genome amplification and high-throughput sequencing to establish the identities of both the host and the attached virus(es). We demonstrate single-cell viral tagging using the faecal microbiome, including cross-tagging of viruses and bacteria between human subjects. A total of 363 unique host-phage pairings were predicted, most of which were subject-specific and involved previously uncharacterized viruses despite the majority of their bacterial hosts having known taxonomy. One-fifth of these pairs were confirmed by multiple individual tagged cells. Viruses targeting more than one bacterial species were conspicuously absent in the host-phage network, suggesting that phages are not major vectors of inter-species horizontal gene transfer in the human gut. A high level of cross-reactivity between phages and bacteria from different subjects was noted despite subject-specific viral profiles, which has implications for faecal micro-biota transplant therapy

Problem Solving Skills Development in Mathematics Teaching

Diplomdarbā „Problēmrisināšanas prasmju attīstīšana matemātikas mācību procesā” tiek noskaidrota skolēnu pieredze problēmu risināšanā, un, domājot par mācību procesa kvalitātes celšanu, tiek izstrādāts skolotāju atbalsta materiāls skolēnu problēmrisināšanas prasmju attīstīšanai matemātikas stundās pamatskolas 7. – 9. klasēs. Darbs sastāv no trīs nodaļām: 1. nodaļā tie apzināti mācību mērķi matemātikā pamatskolā; 2. nodaļā tiek noskaidrota skolēnu pieredze problēmu risināšanā; 3. nodaļā tiek piedāvāti ieteikumi problēmrisināšanas prasmju attīstīšanai. Atslēgvārdi: problēmrisināšanas prasmes, problēmrisināšanas stratēģijas, pētnieciskā mācīšanās, aktīvās mācību metodesThe Diploma Paper “Problem Solving Skills Development in Mathematics Teaching” outlines students' experience in solving of problems. Furthermore, it presents supplementary teachers' material to encourage development of students' problem solving skills via teaching of mathematics. This material is aimed at improving the quality of the teaching process and is intended for Grades 7 – 9 of elementary schools. The Paper consists of three chapters: Chapter I identifies learning objectives in mathematics in elementary schools; Chapter II deals with students' experience in solving problems, while Chapter III contains recommendations for developing of problem solving skills. Keywords: problem solving skills, problem solving strategies, investigative learning, active learning method

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

BackgroundNudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva.ResultsWe recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously.ConclusionsAltogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo. Video Abstract

Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate

The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.This work was funded by grant CP09/00049 Miguel Servet, Instituto de Salud Carlos III, Spain to GD; by projects SAF2009-13032-C02-01 and SAF 2012-31187 (AM), BFU2009-12895-CO2-01 and SAF2010-16240 (FC) from the Spanish Ministry for Science and Innovation (MCINN), FU2008-04501-E from Spanish Ministry for Science and Innovation(MCINN) in the frame of ERA-Net PathoGenoMics and Prometeo/2009/092 from Conselleria D’Educació Generalitat Valenciana,Spain, to AM. MD is recipient of a fellowship from Spanish Ministry of Education FPU2010. MGG was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation (Grant number BES-2008-006029