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Found 35955 matches. Displaying 41-50
Kerner G, Laval G, Patin E, Boisson-Dupuis S, Abel L, Casanova JL, Quintana-Murci L
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Human ancient DNA analyses reveal the high burden of tuberculosis in Europeans over the last 2,000 years

Tuberculosis (TB), usually caused by Mycobacterium tuberculosis bacteria, is the first cause of death from an infectious disease at the worldwide scale, yet the mode and tempo of TB pressure on humans remain unknown. The recent discovery that homozygotes for the P1104A polymorphism of TYK2 are at higher risk to develop clinical forms of TB provided the first evidence of a common, monogenic predisposition to TB, offering a unique opportunity to inform on human co-evolution with a deadly pathogen. Here, we investigate the history of human exposure to TB by determining the evolutionary trajectory of the TYK2 P1104A variant in Europe, where TB is considered to be the deadliest documented infectious disease. Leveraging a large dataset of 1,013 ancient human genomes and using an approximate Bayesian computation approach, we find that the P1104A variant originated in the common ancestors of West Eurasians similar to 30,000 years ago. Furthermore, we show that, following large-scale population movements of Anatolian Neolithic farmers and Eurasian steppe herders into Europe, P1104A has markedly fluctuated in frequency over the last 10,000 years of European history, with a dramatic decrease in frequency after the Bronze Age. Our analyses indicate that such a frequency drop is attributable to strong negative selection starting similar to 2,000 years ago, with a relative fitness reduction on homozygotes of 20%, among the highest in the human genome. Together, our results provide genetic evidence that TB has imposed a heavy burden on European health over the last two millennia.
Mickolajczyk KJ, Shelton PMM, Grasso M, Cao XC, Warrington SE, Aher A, Liu SX, Kapoor TM
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Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

BIOPHYSICAL JOURNAL 2021 MAR 16; 120(6):1020-1030
The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.
Sullivan ZA, Khoury-Hanold W, Lim J, Smillie C, Biton M, Reis BS, Zwick RK, Pope SD, Israni-Winger K, Parsa R, Philip NH, Rashed S, Palm N, Wang A, Mucida D, Regev A, Medzhitov R
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gamma delta T cells regulate the intestinal response to nutrient sensing

SCIENCE 2021 MAR 19; 371(6535):1223-eaba8310 Article eaba8310
The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The "on-demand" induction of this machinery required gamma delta T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of gamma delta T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for gamma delta T cells in nutrient sensing.
Abdalla T, Mansour M, Bouazzi D, Lowes MA, Jemec GBE, Alavi A
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Therapeutic Drug Monitoring in Patients with Suboptimal Response to Adalimumab for Hidradenitis Suppurativa: A Retrospective Case Series

Background Adalimumab, a tumor necrosis factor-alpha inhibitor, is a biologic used for the treatment of moderate-to-severe hidradenitis suppurativa (HS). It is well known that patients may experience loss of efficacy from its use in other conditions, and it is suggested that developing a strategy for therapeutic drug monitoring (TDM) may help secure optimal clinical outcomes. Objectives We sought to determine serum adalimumab concentrations and anti-adalimumab antibody (AAA) status in patients with moderate-to-severe HS. Methods A retrospective case series of 38 patients with suboptimal response to adalimumab 40 mg weekly was conducted at a community dermatology clinic. Adalimumab serum trough levels, AAA status, and inflammatory biomarkers were collected. Blood was drawn on identification of suboptimal response (after a minimum of 12 weeks) and was collected once prior to receiving the next scheduled dose. Kruskal-Wallis and Chi-squared tests were used for data analysis. Results A total of 38 patients had a median adalimumab trough concentration of 8.76 (interquartile range [IQR] 1.3-12.5) mu g/mL. The median duration of adalimumab therapy of all patients was 21 (IQR 12-24) months. AAAs were detected in nine patients (24%), and all had subtherapeutic serum concentrations (< 6 mu g/mL). Patients who were AAA+ had a significantly lower median adalimumab concentration than those who were AAA- (0.02 mu g/mL [range 0.02-0.81] vs. 10.14 [range 0.76-48.00]; p = 0.0006). Conclusion Patients with AAAs had significantly lower serum adalimumab levels. The current study suggests that TDM may identify underlying reasons for suboptimal response and detect patients who may benefit from dose optimization strategies.
Nimmerjahn F, Ravetch JV
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Four keys to unlock IgG

JOURNAL OF EXPERIMENTAL MEDICINE 2021 MAR 1; 218(3):? Article e20201753
The identification of discrete subclasses within the immunoglobulin G (IgG) isotype by Grey and Kunkel (1964. J. Exp. Med. provided the framework for our current understanding of differential IgG subclass activity in protective and self-reactive immune responses.
Olivieri C, Walker C, Karamafrooz A, Wang YJ, Manu VS, Porcelli F, Blumenthal DK, Thomas DD, Bernlohr DA, Sandford SM, Taylor SS, Veglia G
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Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

COMMUNICATIONS BIOLOGY 2021 MAR 10; 4(1):? Article 321
Olivieri, Walker, Karamafrooz et al. show that the fusion of the dynamic J-domain to PKA-C (PKA-CDNAJB1) disrupts the internal allosteric network, attenuating the nucleotide/PKI binding cooperativity. This study suggests that the reduced allosteric cooperativity may contribute to the pathology that PKA-CDNAJB1 drives. An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-C-DNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic alpha subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-C-DNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-C-DNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.
Singer ZS, Ambrose PM, Danino T, Rice CM
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Quantitative measurements of early alphaviral replication dynamics in single cells reveals the basis for superinfection exclusion

CELL SYSTEMS 2021 MAR 17; 12(3):210-219.e3
While decades of research have elucidated many steps of the alphavirus lifecycle, the earliest replication dynamics have remained unclear. This missing time window has obscured early replicase strand-synthesis behavior and prevented elucidation of how the first events of infection might influence subsequent viral competition. Using quantitative live-cell and single-molecule imaging, we observed the initial replicase activity and its strand preferences in situ and measured the trajectory of replication over time. Under this quantitative framework, we investigated viral competition, where one alphavirus is able to exclude superinfection by a second homologous virus. We show that this appears as an indirect phenotypic consequence of a bidirectional competition between the two species, coupled with the rapid onset of viral replication and a limited total cellular carrying capacity. Together, these results emphasize the utility of analyzing viral kinetics within single cells.
Agerer B, Koblischke M, Gudipati V, Montano-Gutierrez LF, Smyth M, Popa A, Genger JW, Endler L, Florian DM, Muhlgrabner V, Graninger M, Aberle SW, Husa AM, Shaw LE, Lercher A, Gattinger P, Torralba-Gombau R, Trapin D, Penz T, Barreca D, Fae I, Wenda S, Traugott M, Walder G, Pickl WF, Thiel V, Allerberger F, Stockinger H, Puchhammer-Stockl E, Weninger W, Fischer G, Hoepler W, Pawelka E, Zoufaly A, Valenta R, Bock C, Paster W, Geyeregger R, Farlik M, Halbritter F, Huppa JB, Aberle JH, Bergthaler A
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SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8(+) T cell responses

SCIENCE IMMUNOLOGY 2021 MAR; 6(57):? Article eabg6461
CD8(+) T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8(+) T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-gamma production and cytotoxic activity of CD8(+) T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8(+) T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8(+) T cell surveillance through point mutations in MHC-I-restricted viral epitopes.
Gaebler C, Falcinelli SD, Stoffel E, Read J, Murtagh R, Oliveira TY, Ramos V, Lorenzi JCC, Kirchherr J, James KS, Allard B, Baker C, Kuruc JD, Caskey M, Archin NM, Siliciano RF, Margolis DM, Nussenzweig MC
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Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA

JOURNAL OF VIROLOGY 2021 MAR; 95(6):? Article e01986-20
The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART) suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.
Wetzel KS, Guerrero-Bustamante CA, Dedrick RM, Ko CC, Freeman KG, Aull HG, Divens AM, Rock JM, Zack KM, Hatfull GF
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CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering

SCIENTIFIC REPORTS 2021 MAR 24; 11(1):? Article 6796
Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.