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April 21, 2020  |  

A systematic review of the Trypanosoma cruzi genetic heterogeneity, host immune response and genetic factors as plausible drivers of chronic chagasic cardiomyopathy.

Chagas disease is a complex tropical pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite displays massive genetic diversity and has been classified by international consensus in at least six Discrete Typing Units (DTUs) that are broadly distributed in the American continent. The main clinical manifestation of the disease is the chronic chagasic cardiomyopathy (CCC) that is lethal in the infected individuals. However, one intriguing feature is that only 30-40% of the infected individuals will develop CCC. Some authors have suggested that the immune response, host genetic factors, virulence factors and even the massive genetic heterogeneity of T. cruzi are responsible of this clinical pattern. To date, no conclusive data support the reason why a few percentages of the infected individuals will develop CCC. Therefore, we decided to conduct a systematic review analysing the host genetic factors, immune response, cytokine production, virulence factors and the plausible association of the parasite DTUs and CCC. The epidemiological and clinical implications are herein discussed.

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April 21, 2020  |  

Vaccine-induced protection from homologous tier 2 SHIV challenge in nonhuman primates depends on serum-neutralizing antibody titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ~1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

Despite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.We have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.We found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10-7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.Our findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.

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April 21, 2020  |  

Rapid and Focused Maturation of a VRC01-Class HIV Broadly Neutralizing Antibody Lineage Involves Both Binding and Accommodation of the N276-Glycan.

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ~24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

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April 21, 2020  |  

Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes. Copyright © 2019 Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Mutation of a bHLH transcription factor allowed almond domestication.

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F1 population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase-encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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April 21, 2020  |  

Genome assembly of a tropical maize inbred line provides insights into structural variation and crop improvement.

Maize is one of the most important crops globally, and it shows remarkable genetic diversity. Knowledge of this diversity could help in crop improvement; however, gold-standard genomes have been elucidated only for modern temperate varieties. Here, we present a high-quality reference genome (contig N50 of 15.78?megabases) of the maize small-kernel inbred line, which is derived from a tropical landrace. Using haplotype maps derived from B73, Mo17 and SK, we identified 80,614 polymorphic structural variants across 521 diverse lines. Approximately 22% of these variants could not be detected by traditional single-nucleotide-polymorphism-based approaches, and some of them could affect gene expression and trait performance. To illustrate the utility of the diverse SK line, we used it to perform map-based cloning of a major effect quantitative trait locus controlling kernel weight-a key trait selected during maize improvement. The underlying candidate gene ZmBARELY ANY MERISTEM1d provides a target for increasing crop yields.

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April 21, 2020  |  

Reduced frequency of HIV superinfection in a high-risk cohort in Zambia.

Rates of HIV-1 superinfection, re-infection with a genetically distinct virus despite HIV-1 specific immune responses, vary in different risk populations. We previously found the rates of superinfection were similar to primary HIV infection (PHI) in a Zambian heterosexual transmission cohort. Here, we conduct a similar analysis of 47 HIV-positive Zambians from an acute infection cohort with more frequent follow-up, all infected by non-spousal partners. We identified only one case of superinfection in the first two years, significantly fewer than in our previous study, which was likely due to increased counseling during acute infection and an overall population-wide decline in factors associated with HIV transmission. The predominant virus detected after superinfection was a recombinant of the transmitted founder (TF) and the superinfecting strain. The superinfected individual mounted a neutralizing antibody response to the primary TF virus, which remained TF-specific over time and even after superinfection, did not neutralize the superinfecting variant.Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020  |  

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

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April 21, 2020  |  

Genetic basis of functional variability in adhesion G protein-coupled receptors.

The enormous sizes of adhesion G protein-coupled receptors (aGPCRs) go along with complex genomic exon-intron architectures giving rise to multiple mRNA variants. There is a need for a comprehensive catalog of aGPCR variants for proper evaluation of the complex functions of aGPCRs found in structural, in vitro and animal model studies. We used an established bioinformatics pipeline to extract, quantify and visualize mRNA variants of aGPCRs from deeply sequenced transcriptomes. Data analysis showed that aGPCRs have multiple transcription start sites even within introns and that tissue-specific splicing is frequent. On average, 19 significantly expressed transcript variants are derived from a given aGPCR gene. The domain architecture of the N terminus encoded by transcript variants often differs and N termini without or with an incomplete seven-helix transmembrane anchor as well as separate seven-helix transmembrane domains are frequently derived from aGPCR genes. Experimental analyses of selected aGPCR transcript variants revealed marked functional differences. Our analysis has an impact on a rational design of aGPCR constructs for structural analyses and gene-deficient mouse lines and provides new support for independent functions of both, the large N terminus and the transmembrane domain of aGPCRs.

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April 21, 2020  |  

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

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April 21, 2020  |  

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

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April 21, 2020  |  

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity.

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

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