Lifelong Imaging Surveillance is Indicated for Patients with Primary Retroperitoneal Liposarcoma.

BACKGROUND: Surveillance imaging of patients with retroperitoneal liposarcoma (RP-LPS) after surgical resection is based on a projected risk of locoregional and distant recurrence. The duration of surveillance is not well defined because the natural history of RP-LPS after treatment is poorly understood. This study evaluated the long-term risk of recurrence and disease-specific survival (DSS) for a cohort of patients with at least 10 years of progression-free survival (10yr-PFS) from their primary resection. METHODS: The prospective University of California, Los Angeles (UCLA) Sarcoma Database identified RP-LPS patients with 10yr-PFS after initial resection. The patients in the 10yr-PFS cohort were subsequently evaluated for recurrence and DSS. The time intervals start at date of initial surgical resection. Cox proportional hazards models were used to determine factors associated with recurrence and DSS. RESULTS: From 1972 to 2010, 76 patients with RP-LPS had at least 10 years of follow-up evaluation. Of these 76 patients, 39 (51%) demonstrated 10yr-PFS. The median follow-up period was 15 years (range 10-33 years). Among the 10yr-PFS patients, 49% (19/39) experienced a recurrence at least 10 years after surgery. Of those who experienced recurrence, 42% (8/19) died of disease. Neither long-term recurrence nor DSS were significantly associated with age, sex, tumor size, LPS subtype, surgical margin, or perioperative treatment with radiation or chemotherapy. CONCLUSION: Patients who have primary RP-LPS treated with surgical resection ± multimodality therapy face a long-term risk of recurrence and disease-specific death unacknowledged by current surveillance imaging guidelines. Among the patients with 10yr-PFS, 49% experienced a recurrence, and 42% of those died of disease. These findings suggest a need for lifelong surveillance imaging for patients with RP-LPS.

Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae.

Escherichia coli and other Enterobacteriaceae are diverse species with "open" pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553 Escherichia and 274 non-Escherichia spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.

Genomic network analysis of environmental and livestock F-type plasmid populations.

F-type plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum ß-lactamases, particularly in Enterobacterales. Organising this plasmid diversity is challenging, and current knowledge is largely based on plasmids from clinical settings. Here, we present a network community analysis of a large survey of F-type plasmids from environmental (influent, effluent and upstream/downstream waterways surrounding wastewater treatment works) and livestock settings. We use a tractable and scalable methodology to examine the relationship between plasmid metadata and network communities. This reveals how niche (sampling compartment and host genera) partition and shape plasmid diversity. We also perform pangenome-style analyses on network communities. We show that such communities define unique combinations of core genes, with limited overlap. Building plasmid phylogenies based on alignments of these core genes, we demonstrate that plasmid accessory function is closely linked to core gene content. Taken together, our results suggest that stable F-type plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The association of F-type plasmids with AMR may reflect their suitability for rapid niche adaptation.

Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes.

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.

Contrasting community assembly processes structure lotic bacteria metacommunities along the river continuum.

The heterogeneous nature of lotic habitats plays an important role in the complex ecological and evolutionary processes that structure the microbial communities within them. Due to such complexity, our understanding of lotic microbial ecology still lacks conceptual frameworks for the ecological processes that shape these communities. We explored how bacterial community composition and underlying ecological assembly processes differ between lotic habitats by examining community composition and inferring community assembly processes across four major habitat types (free-living, particle-associated, biofilm on benthic stones and rocks, and sediment). This was conducted at 12 river sites from headwater streams to the main river in the River Thames, UK. Our results indicate that there are distinct differences in the bacterial communities between four major habitat types, with contrasting ecological processes shaping their community assembly processes. While the mobile free-living and particle-associated communities were consistently less diverse than the fixed sediment and biofilm communities, the latter two communities displayed higher homogeneity across the sampling sites. This indicates that the relative influence of deterministic environmental filtering is elevated in sediment and biofilm communities compared with free-living and particle-associated communities, where stochastic processes play a larger role.

Robust neutralizing antibodies to SARS-CoV-2 infection persist for months.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic with millions infected and more than 1 million fatalities. Questions regarding the robustness, functionality, and longevity of the antibody response to the virus remain unanswered. Here, on the basis of a dataset of 30,082 individuals screened at Mount Sinai Health System in New York City, we report that the vast majority of infected individuals with mild-to-moderate COVID-19 experience robust immunoglobulin G antibody responses against the viral spike protein. We also show that titers are relatively stable for at least a period of about 5 months and that anti-spike binding titers significantly correlate with neutralization of authentic SARS-CoV-2. Our data suggest that more than 90% of seroconverters make detectable neutralizing antibody responses. These titers remain relatively stable for several months after infection.

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes.

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

Forty years of reform and opening up: China's progress toward a sustainable path.

After 40 years of reform and "opening up," China has made remarkable economic progress. Such economic prosperity, however, has been coupled with environmental degradation. We analyze diverse long-term data to determine whether China is experiencing a decoupling of economic growth and environmental impacts, and where China stands with respect to the Sustainable Development Goals (SDGs) in terms of reducing regional division, urban-rural gap, social inequality, and land-based impacts on oceans. The results highlight that China's desire to achieve "ecological civilization" has resulted in a decoupling trend for major pollutants since 2015, while strong coupling remains with CO2 emissions. Progress has been made in health care provision, poverty reduction, and gender equity in education, while income disparity continues between regions and with rural-urban populations. There is a considerable way to go toward achieving delivery of the SDGs; however, China's progress toward economic prosperity and concomitant sustainability provides important insights for other countries.

Extending the Stalk Enhances Immunogenicity of the Influenza Virus Neuraminidase.

Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when the two glycoproteins are closely associated. To address this issue, here we investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild-type NA or the stalk-extended NA variants were used to vaccinate mice. The virus with the 30-amino-acid stalk extension induced significantly higher anti-NA IgG responses (characterized by increased in vitro antibody-dependent cellular cytotoxicity [ADCC] activity) than the wild-type PR8 virus, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. On the basis of these results, we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that its height surpasses that of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA.IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts toward more effective influenza virus vaccines.

A mosaic hemagglutinin-based influenza virus vaccine candidate protects mice from challenge with divergent H3N2 strains.

Current seasonal influenza virus vaccines only provide limited, short-lived protection, and antigenic drift in the hemagglutinin surface glycoprotein necessitates their annual re-formulation and re-administration. To overcome these limitations, universal vaccine strategies that aim at eliciting broadly protective antibodies to conserved epitopes of the hemagglutinin show promise for protecting against diverse and drifted influenza viruses. Here a vaccination strategy that focuses antibody responses to conserved epitopes of the H3 hemagglutinin is described. The approach is based on antigenic silencing of the immunodominant major antigenic sites of an H3 protein from 2014 by replacing them with corresponding sequences of exotic avian hemagglutinins, yielding "mosaic" hemagglutinins. In mice, vaccination with inactivated viruses expressing mosaic hemagglutinins induced highly cross-reactive antibodies against the H3 stalk domain that elicited Fc-mediated effector functions in vitro. In addition, the mosaic viruses elicited head-specific antibodies with neutralizing and hemagglutination-inhibiting activity against recent H3N2 viruses in vitro. Immune sera protected mice from heterologous challenge with viruses carrying H3 proteins from 1968 and 1982, whereas immune sera generated with a seasonal vaccine did not protect. Consequently, the mosaic vaccination approach provides a promising avenue toward a universal influenza virus vaccine.

Human Monoclonal Antibodies Potently Neutralize Zika Virus and Select for Escape Mutations on the Lateral Ridge of the Envelope Protein.

The mosquito-borne Zika virus (ZIKV) has been causing epidemic outbreaks on a global scale. Virus infection can result in severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Here, we characterized monoclonal antibodies isolated from a patient with an active Zika virus infection that potently neutralized virus infection in Vero cells at the nanogram-per-milliliter range. In addition, these antibodies enhanced internalization of virions into human leukemia K562 cells in vitro, indicating their possible ability to cause antibody-dependent enhancement of disease. Escape variants of the ZIKV MR766 strain to a potently neutralizing antibody, AC10, exhibited an amino acid substitution at residue S368 in the lateral ridge region of the envelope protein. Analysis of publicly availably ZIKV sequences revealed the S368 site to be conserved among the vast majority (97.6%) of circulating strains. We validated the importance of this residue by engineering a recombinant virus with an S368R point mutation that was unable to be fully neutralized by AC10. Four out of the 12 monoclonal antibodies tested were also unable to neutralize the virus with the S368R mutation, suggesting this region to be an important immunogenic epitope during human infection. Last, a time-of-addition infection assay further validated the escape variant and showed that all monoclonal antibodies inhibited virus binding to the cell surface. Thus, the present study demonstrates that the lateral ridge region of the envelope protein is likely an immunodominant, neutralizing epitope.IMPORTANCE Zika virus (ZIKV) is a global health threat causing severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Here, we analyzed the human monoclonal antibody response to acute ZIKV infection and found that neutralizing antibodies could not elicit Fc-mediated immune effector functions but could potentiate antibody-dependent enhancement of disease. We further identified critical epitopes involved with neutralization by generating and characterizing escape variants by whole-genome sequencing. We demonstrate that the lateral ridge region, particularly the S368 amino acid site, is critical for neutralization by domain III-specific antibodies.

Antibodies Elicited by an NS1-Based Vaccine Protect Mice against Zika Virus.

Zika virus is a mosquito-borne flavivirus which can cause severe disease in humans, including microcephaly and other congenital malformations in newborns and Guillain-Barré syndrome in adults. There are currently no approved prophylactics or therapeutics for Zika virus; the development of a safe and effective vaccine is an urgent priority. Preclinical studies suggest that the envelope glycoprotein can elicit potently neutralizing antibodies. However, such antibodies are implicated in the phenomenon of antibody-dependent enhancement of disease. We have previously shown that monoclonal antibodies targeting the Zika virus nonstructural NS1 protein are protective without inducing antibody-dependent enhancement of disease. Here, we investigated whether the NS1 protein itself is a viable vaccine target. Wild-type mice were vaccinated with an NS1-expressing DNA plasmid followed by two adjuvanted protein boosters, which elicited high antibody titers. Passive transfer of the immune sera was able to significantly protect STAT2 knockout mice against lethal challenge by Zika virus. In addition, long-lasting NS1-specific IgG responses were detected in serum samples from patients in either the acute or the convalescent phase of Zika virus infection. These NS1-specific antibodies were able to functionally engage Fcγ receptors. In contrast, envelope-specific antibodies did not activate Fc-mediated effector functions on infected cells. Our data suggest that the Zika virus NS1 protein, which is expressed on infected cells, is critical for Fc-dependent cell-mediated immunity. The present study demonstrates that the Zika virus NS1 protein is highly immunogenic and can elicit protective antibodies, underscoring its potential for an effective Zika virus vaccine.IMPORTANCE Zika virus is a global public health threat that causes microcephaly and congenital malformations in newborns and Guillain-Barré syndrome in adults. Currently, no vaccines or treatments are available. While antibodies targeting the envelope glycoprotein can neutralize virus, they carry the risk of antibody-dependent enhancement of disease (ADE). In contrast, antibodies generated against the NS1 protein can be protective without eliciting ADE. The present study demonstrates the effectiveness of an NS1-based vaccine in eliciting high titers of protective antibodies against Zika virus disease in a mouse model. Sera generated by this vaccine can elicit Fc-mediated effector functions against Zika virus-infected cells. Lastly, we provide human data suggesting that the antibody response against the Zika virus NS1 protein is long-lasting and functionally active. Overall, our work will inform the development of a safe and effective Zika virus vaccine.

Soil parameters, land use, and geographical distance drive soil bacterial communities along a European transect.

To better understand the relationship between soil bacterial communities, soil physicochemical properties, land use and geographical distance, we considered for the first time ever a European transect running from Sweden down to Portugal and from France to Slovenia. We investigated 71 sites based on their range of variation in soil properties (pH, texture and organic matter), climatic conditions (Atlantic, alpine, boreal, continental, Mediterranean) and land uses (arable, forest and grassland). 16S rRNA gene amplicon pyrosequencing revealed that bacterial communities highly varied in diversity, richness, and structure according to environmental factors. At the European scale, taxa area relationship (TAR) was significant, supporting spatial structuration of bacterial communities. Spatial variations in community diversity and structure were mainly driven by soil physicochemical parameters. Within soil clusters (k-means approach) corresponding to similar edaphic and climatic properties, but to multiple land uses, land use was a major driver of the bacterial communities. Our analyses identified specific indicators of land use (arable, forest, grasslands) or soil conditions (pH, organic C, texture). These findings provide unprecedented information on soil bacterial communities at the European scale and on the drivers involved; possible applications for sustainable soil management are discussed.

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples.

BACKGROUND: Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, 'ResPipe'. RESULTS: Taxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve < 1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment. CONCLUSIONS: Sequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available ( https://gitlab.com/hsgweon/ResPipe ).

Human antibodies targeting Zika virus NS1 provide protection against disease in a mouse model.

Zika virus is a mosquito-borne flavivirus closely related to dengue virus that can cause severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Specific treatments and vaccines for Zika virus are not currently available. Here, we isolate and characterize four monoclonal antibodies (mAbs) from an infected patient that target the non-structural protein NS1. We show that while these antibodies are non-neutralizing, NS1-specific mAbs can engage FcγR without inducing antibody dependent enhancement (ADE) of infection in vitro. Moreover, we demonstrate that mAb AA12 has protective efficacy against lethal challenges of African and Asian lineage strains of Zika virus in Stat2-/- mice. Protection is Fc-dependent, as a mutated antibody unable to activate known Fc effector functions or complement is not protective in vivo. This study highlights the importance of the ZIKV NS1 protein as a potential vaccine antigen.

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-FcγR. Using this method, the contribution of specific Fc-FcγR interactions conferred by immunoglobulins can be determined using an in vitro assay.

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.