Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism

<p>Abstract</p> <p>Background</p> <p>Evolution of unisexual flowers entails one of the most extreme changes in plant development. Cultivated spinach, <it>Spinacia oleracea </it>L., is uniquely suited for the study of unisexual flower development as it is dioecious and it achieves unisexually by the absence of organ development, rather than by organ abortion or suppression. Male staminate flowers lack fourth whorl primordia and female pistillate flowers lack third whorl primordia. Based on theoretical considerations, early inflorescence or floral organ identity genes would likely be directly involved in sex-determination in those species in which organ initiation rather than organ maturation is regulated. In this study, we tested the hypothesis that sexual dimorphism occurs through the regulation of B class floral organ gene expression by experimentally knocking down gene expression by viral induced gene silencing.</p> <p>Results</p> <p>Suppression of B class genes in spinach resulted in the expected homeotic transformation of stamens into carpels but also affected the number of perianth parts and the presence of fourth whorl. Phenotypically normal female flowers developed on <it>SpPI</it>-silenced male plants. Suppression of the spinach C class floral organ identity gene, <it>SpAG</it>, resulted in loss of reproductive organ identity, and indeterminate flowers, but did not result in additional sex-specific characteristics or structures. Analysis of the genomic sequences of both <it>SpAP3 </it>and <it>SpPI </it>did not reveal any allelic differences between males and females.</p> <p>Conclusion</p> <p>Sexual dimorphism in spinach is not the result of homeotic transformation of established organs, but rather is the result of differential initiation and development of the third and fourth whorl primordia. <it>SpAG </it>is inferred to have organ identity and meristem termination functions similar to other angiosperm C class genes. In contrast, while <it>SpPI </it>and <it>SpAP3 </it>resemble other angiosperms in their essential functions in establishing stamen identity, they also appear to have an additional function in regulating organ number and identity outside of the third whorl. We present a model for the evolution of dioecy in spinach based on the regulation of B class expression.</p

Development of a gene silencing DNA vector derived from a broad host range geminivirus

<p>Abstract</p> <p>Background</p> <p>Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.</p> <p>Results</p> <p>The construction of a gene silencing vector derived from the geminivirus <it>Beet curly top virus </it>(BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (<it>rbcS</it>), <it>transketolase</it>, the sulfur allele of magnesium chelatase (<it>ChlI</it>), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.</p> <p>Conclusion</p> <p>The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.</p

Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus.

Funder: open philanthropy projectFunder: jpb foundationVaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model

A Tandem Mass Spectrometry Sequence Database Search Method for Identification of O-Fucosylated Proteins by Mass Spectrometry.

Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/ z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to reanalyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins

Restricting HIV-1 pathways for escape using rationally designed anti–HIV-1 antibodies

Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46G54W, which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46^(G54W) variants designed using analyses of the NIH45-46–gp120 complex structure and sequences of NIH45-46^(G54W)–resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti–HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2–gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46G54W arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1_(YU-2)–infected humanized mice, with viremic control exhibited when a third antibody, 10–1074, was added to the combination

Broad Neutralization by a Combination of Antibodies Recognizing the CD4 Binding Site and a New Conformational Epitope on the HIV-1 Envelope Protein

Two to three years after infection, a fraction of HIV-1–infected individuals develop serologic activity that neutralizes most viral isolates. Broadly neutralizing antibodies that recognize the HIV-1 envelope protein have been isolated from these patients by single-cell sorting and by neutralization screens. Here, we report a new method for anti–HIV-1 antibody isolation based on capturing single B cells that recognize the HIV-1 envelope protein expressed on the surface of transfected cells. Although far less efficient than soluble protein baits, the cell-based capture method identified antibodies that bind to a new broadly neutralizing epitope in the vicinity of the V3 loop and the CD4-induced site (CD4i). The new epitope is expressed on the cell surface form of the HIV-1 spike, but not on soluble forms of the same envelope protein. Moreover, the new antibodies complement the neutralization spectrum of potent broadly neutralizing anti-CD4 binding site (CD4bs) antibodies obtained from the same individual. Thus, combinations of potent broadly neutralizing antibodies with complementary activity can account for the breadth and potency of naturally arising anti–HIV-1 serologic activity. Therefore, vaccines aimed at eliciting anti–HIV-1 serologic breadth and potency should not be limited to single epitopes

Characteristics of the Earliest Cross-Neutralizing Antibody Response to HIV-1

Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%–30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2–3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs