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Berlin Brandenburg

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  • 1
    In: PLoS ONE, 2015, Vol.10(1)
    Description: Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids.
    Keywords: Research Article
    E-ISSN: 1932-6203
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  • 2
    In: Nature, 2017
    Description: In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.
    Keywords: Blood Donors ; Mutagenesis, Insertional ; Antibodies, Protozoan -- Chemistry ; Antigens, Protozoan -- Immunology ; Malaria -- Immunology ; Plasmodium Falciparum -- Immunology ; Receptors, Immunologic -- Genetics;
    ISSN: 0028-0836
    E-ISSN: 1476-4687
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  • 3
    Language: English
    In: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, July 2013, Vol.57(1), pp.40-7
    Description: In experimental models of human and mouse malaria, sterilizing liver stage immunity that blocks progression of Plasmodium infection to the symptomatic blood stage can be readily demonstrated. However, it remains unclear whether individuals in malaria-endemic areas acquire such immunity. In Mali, 251 healthy children and adults aged 4-25 years who were free of blood-stage Plasmodium infection by polymerase chain reaction (PCR) were enrolled in a longitudinal study just prior to an intense 6-month malaria season. Subsequent clinical malaria episodes were detected by weekly active surveillance and self-referral. Asymptomatic P. falciparum infections were detected by blood-smear microscopy and PCR analysis of dried blood spots that had been collected every 2 weeks for 7 months. As expected, the risk of clinical malaria decreased with increasing age (log-rank test, P = .0038). However, analysis of PCR data showed no age-related differences in P. falciparum infection risk (log-rank test, P = .37). Despite years of exposure to intense P. falciparum transmission, there is no evidence of acquired, sterile immunity to P. falciparum infection in this population, even as clinical immunity to blood-stage malaria is clearly acquired. Understanding why repeated P. falciparum infections do not induce sterile protection may lead to insights for developing vaccines that target the liver stage in malaria-endemic populations.
    Keywords: Plasmodium Falciparum ; Endemic Population ; Infection Risk ; Malaria ; Preerythrocytic Immunity ; Adaptive Immunity ; Malaria, Falciparum -- Epidemiology ; Plasmodium Falciparum -- Immunology
    ISSN: 10584838
    E-ISSN: 1537-6591
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  • 4
    Language: English
    In: Trends in Parasitology, June 2012, Vol.28(6), pp.248-257
    Description: malaria remains a global public health threat. Optimism that a highly effective malaria vaccine can be developed stems in part from the observation that humans can acquire immunity to malaria through experimental and natural infection. Recent advances in systems immunology could accelerate efforts to unravel the mechanisms of acquired immunity to malaria. Here, we review the tools of systems immunology, their current limitations in the context of human malaria research, and the human ‘models’ of malaria immunity to which these tools can be applied.
    Keywords: Systems Immunology ; Malaria ; Plasmodium Falciparum ; Biology ; Veterinary Medicine
    ISSN: 1471-4922
    E-ISSN: 1471-5007
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  • 5
    Language: English
    In: PloS one, 2015, Vol.10(4), pp.e0125090
    Description: Vaccine-induced immunity depends on long-lived plasma cells (LLPCs) that maintain antibody levels. A recent mouse study showed that Plasmodium chaubaudi infection reduced pre-existing influenza-specific antibodies--raising concerns that malaria may compromise pre-existing vaccine responses. We extended these findings to P. yoelii infection, observing decreases in antibodies to model antigens in inbred mice and to influenza in outbred mice, associated with LLPC depletion and increased susceptibility to influenza rechallenge. We investigated the implications of these findings in Malian children by measuring vaccine-specific IgG (tetanus, measles, hepatitis B) before and after the malaria-free 6-month dry season, 10 days after the first malaria episode of the malaria season, and after the subsequent dry season. On average, vaccine-specific IgG did not decrease following acute malaria. However, in some children malaria was associated with an accelerated decline in vaccine-specific IgG, underscoring the need to further investigate the impact of malaria on pre-existing vaccine-specific antibodies.
    Keywords: Antibodies, Protozoan -- Immunology ; Antibodies, Viral -- Immunology ; Antigens, Protozoan -- Immunology ; Antigens, Viral -- Immunology ; Malaria -- Immunology ; Malaria Vaccines -- Immunology
    E-ISSN: 1932-6203
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 09 June 2015, Vol.112(23), pp.7159-64
    Description: Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.
    Keywords: Droplet-Based Microfluidics ; High-Throughput DNA Sequencing ; Protein Engineering ; Microfluidic Analytical Techniques ; Mutation ; Glycoside Hydrolases -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 7
    Language: English
    In: PLoS Computational Biology, 2010, Vol.6(1), p.e1000647
    Description: We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved. ; The β2 adrenergic receptor (β2AR) is involved in regulating many cellular processes such as smooth muscle relaxation in the airways and the vasculature. Drugs that activate the β2AR are used in treating asthma and chronic obstructive pulmonary disorder (COPD), and prolonged use of these drugs leads to the loss of their effects. Thus, a dynamic model of how the β2AR responds to different drugs is fundamental to their rational use. In this study a consensus model of G protein coupled receptor kinase (GRK)-mediated receptor regulation was formulated based on quantitative measures of six processes involved in β2AR regulation. This model was then used to simulate the consequences of manipulating key rates associated with the GRK-mediated β2AR regulation, leading to predictions which will provide a useful framework for further tests and elaborations of the model in basic and clinical research.
    Keywords: Research Article ; Biochemistry -- Cell Signaling And Trafficking Structures ; Biochemistry -- Theory And Simulation ; Cell Biology -- Cell Signaling ; Pharmacology ; Physiology -- Cell Signaling ; Respiratory Medicine -- Asthma
    ISSN: 1553-734X
    E-ISSN: 1553-7358
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  • 8
    Language: English
    In: Malaria journal, 04 October 2014, Vol.13, pp.393
    Description: As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material. The density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared. Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson's r=0.58, P〈0.001) and symptomatic (Pearson's r=0.70, P〈0.0001) P. falciparum infections. Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.
    Keywords: DNA, Protozoan -- Blood ; Dried Blood Spot Testing -- Methods ; Malaria, Falciparum -- Diagnosis ; Plasmodium Falciparum -- Genetics ; Real-Time Polymerase Chain Reaction -- Methods
    E-ISSN: 1475-2875
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  • 9
    Language: English
    In: Cell Reports, 13 October 2015, Vol.13(2), pp.425-439
    Description: Malaria-specific antibody responses are short lived in children, leaving them susceptible to repeated bouts of febrile malaria. The cellular and molecular mechanisms underlying this apparent immune deficiency are poorly understood. Recently, T follicular helper (Tfh) cells have been shown to play a critical role in generating long-lived antibody responses. We show that Malian children have resting PD-1 CXCR5 CD4 Tfh cells in circulation that resemble germinal center Tfh cells phenotypically and functionally. Within this population, PD-1 CXCR5 CXCR3 Tfh cells are superior to Th1-polarized PD-1 CXCR5 CXCR3 Tfh cells in helping B cells. Longitudinally, we observed that malaria drives Th1 cytokine responses, and accordingly, the less-functional Th1-polarized Tfh subset was preferentially activated and its activation did not correlate with antibody responses. These data provide insights into the Tfh cell biology underlying suboptimal antibody responses to malaria in children and suggest that vaccine strategies that promote CXCR3 Tfh cell responses may improve malaria vaccine efficacy. Malaria-specific antibody responses are short lived in children, leaving them susceptible to repeated malaria infections. Tfh cells are critical for long-lived antibody responses. Obeng-Adjei et al. provide evidence that malaria activates less-functional Th1-like PD-1 CXCR5 CXCR3 Tfh cells that are disassociated from B cell/antibody responses.
    Keywords: Biology
    ISSN: 2211-1247
    E-ISSN: 2211-1247
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  • 10
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 20 October 2015, Vol.112(42), pp.13075-80
    Description: The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.
    Keywords: Cd8+ T Cells ; Don ; Adjunctive Therapy ; Cerebral Malaria ; Glutamine Metabolism ; Antimalarials -- Therapeutic Use ; Diazooxonorleucine -- Therapeutic Use ; Glutamine -- Metabolism ; Malaria, Cerebral -- Drug Therapy ; Malaria, Falciparum -- Drug Therapy
    ISSN: 00278424
    E-ISSN: 1091-6490
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