New avenues for potentially seeking microbial responses to climate change beneath Antarctic ice shelves.

The signs of climate change are undeniable, and the impact of these changes on ecosystem function heavily depends on the response of microbes that underpin the food web. Antarctic ice shelf is a massive mass of floating ice that extends from the continent into the ocean, exerting a profound influence on global carbon cycles. Beneath Antarctic ice shelves, marine ice stores valuable genetic information, where marine microbial communities before the industrial revolution are archived. Here, in this proof-of-concept, by employing a combination of single-cell technologiesand metagenomics, we have been able to sequence frozen microbial DNA (≈300 years old) stored in the marine ice core B15 collected from the Filchnner-Ronne Ice Shelf. Metagenomic data indicated that Proteobacteria and Thaumarchaeota (e.g., Nitrosopumilus spp.), followed by Actinobacteria (e.g., Actinomarinales), were abundant. Remarkably, our data allow us to "travel to the past" and calibrate genomic and genetic evolutionary changes for ecologically relevant microbes and functions, such as Nitrosopumilus spp., preserved in the marine ice (≈300 years old) with those collected recently in seawater under an ice shelf (year 2017). The evolutionary divergence for the ammonia monooxygenase gene amoA involved in chemolithoautotrophy was about 0.88 amino acid and 2.8 nucleotide substitution rate per 100 sites in a century, while the accumulated rate of genomic SNPs was 2,467 per 1 Mb of genome and 100 years. Whether these evolutionary changes remained constant over the last 300 years or accelerated during post-industrial periods remains an open question that will be further elucidated. IMPORTANCE: Several efforts have been undertaken to predict the response of microbes under climate change, mainly based on short-term microcosm experiments under forced conditions. A common concern is that manipulative experiments cannot properly simulate the response of microbes to climate change, which is a long-term evolutionary process. In this proof-of-concept study with a limited sample size, we demonstrate a novel approach yet to be fully explored in science for accessing genetic information from putative past marine microbes preserved under Antarctic ice shelves before the industrial revolution. This potentially allows us estimating evolutionary changes as exemplified in our study. We advocate for gathering a more comprehensive Antarctic marine ice core data sets across various periods and sites. Such a data set would enable the establishment of a robust baseline, facilitating a better assessment of the potential effects of climate change on key genetic signatures of microbes.

[Coronary lithotripsy in calcified lesions: fragmented calcium]. / Litotricia coronaria en lesiones calcificadas: fragmentando el calcio.

Background: Complex calcified coronary lesions are a frequent finding during percutaneous coronary intervention, representing for decades a challenge and limitation in patients with indication of revascularization, due to suboptimal angiographic results, high incidence of perioperative complications and long-term adverse events despite the multiple strategies employed, such as the use of cutting balloon, high-pressure balloons or rotational or orbital atherectomy, interventions with limitations that have hindered its routine use, recently a new plaque modification technique known as coronary intravascular lithotripsy has burst into the treatment of this complex entity, which consists in the use of a specially modified balloon for the emission of pulsatile mechanical energy (sonic pressure waves) that allows modifying the calcified plate. Clinical case: By presenting a series of clinical cases and reviewing the literature, our initial experience is presented, key elements are summarized and discussed in the understanding of this new intervention technique necessary for decision making. Conclusion: Coronary intravascular lithotripsy is projected as a promising technique for the modification and preparation of superficial and deep calcified coronary lesions, through microfractures that allow the apposition and effective expansion of the stent, strategy that according to different trials (Disrupt CAD series, SOLSTICE assay) and records presents a high efficiency and good safety profile, data consistent with our initial experience.

Introducción: las lesiones coronarias calcificadas complejas son un hallazgo frecuente durante el intervencionismo coronario percutáneo, han representado durante décadas un desafío y limitante en pacientes con indicación de revascularización, debido a resultados angiográficos subóptimos, alta incidencia de complicaciones perioperatorias y eventos adversos a largo plazo a pesar de las múltiples estrategias empleadas, como el uso de balones de corte, balones de alta presión o la aterectomía rotacional u orbital, intervenciones con limitantes que han dificultado su uso rutinario. Recientemente, una nueva técnica de modificación de placa conocida como litotricia intravascular coronaria ha irrumpido en el tratamiento de esta compleja entidad, la cual consiste en la utilización de un balón especialmente modificado para la emisión de energía mecánica pulsátil (ondas de presión sónicas) que permite modificar la placa calcificada. Caso clínico: mediante la presentación de una serie de casos clínico y revisión de literatura se presenta nuestra experiencia inicial, se resume y discuten elementos claves en el entendimiento de esta nueva técnica de intervencionismo necesarios para la toma de decisiones. Conclusión: la litotricia intravascular coronaria se proyecta como una técnica prometedora para la modificación y preparación de lesiones coronarias calcificadas superficiales y profundas, mediante microfracturas que permiten la aposición y expansión efectiva del stent; estrategia que de acuerdo con diferentes ensayos (serie Disrupt CAD, ensayo SOLSTICE) y registros presenta una eficacia alta y buen perfil de seguridad, datos concordantes con nuestra experiencia inicial.

Novel RNA viruses from the Atlantic Ocean: Ecogenomics, biogeography, and total virioplankton mass contribution from surface to the deep ocean.

Marine viruses play a major role in the energy and nutrient cycle and affect the evolution of their hosts. Despite their importance, there is still little knowledge about RNA viruses. Here, we have explored the Atlantic Ocean, from surface to deep (4.296 m), and used viromics and quantitative methods to unveil the genomics, biogeography, and the mass contribution of RNA viruses to the total viroplankton. A total of 2481 putative RNA viral contigs (>500 bp) and 107 larger bona fide RNA viral genomes (>2.5 kb) were identified; 88 of them representing novel viruses belonging mostly to two clades: Yangshan assemblage (sister clade to the class Alsuviricetes) and Nodaviridae. These viruses were highly endemic and locally abundant, with little or no presence in other oceans since only ≈15% of them were found in at least one of the Tara sampling metatranscriptomes. Quantitative data indicated that the abundance of RNA viruses in the surface and deep chlorophyll maximum zone was within ≈106 VLP/mL representing a potential contribution of 5.2%-24.4% to the total viroplankton community (DNA and RNA viruses), with DNA viruses being the predominant members (≈107 VLP/mL). However, for the deep sample, the observed trend was the opposite, although as further discussed, several biases should be considered. Together these results contribute to our understanding of the diversity, abundance, and distribution of RNA viruses in the oceans and provide a basis for further investigation into their ecological roles and biogeography.

Into the Dark: Exploring the Deep Ocean with Single-Virus Genomics.

Single-virus genomics (SVGs) has been successfully applied to ocean surface samples allowing the discovery of widespread dominant viruses overlooked for years by metagenomics, such as the uncultured virus vSAG 37-F6 infecting the ubiquitous Pelagibacter spp. In SVGs, one uncultured virus at a time is sorted from the environmental sample, whole-genome amplified, and sequenced. Here, we have applied SVGs to deep-ocean samples (200-4000 m depth) from global Malaspina and MEDIMAX expeditions, demonstrating the feasibility of this method in deep-ocean samples. A total of 1328 virus-like particles were sorted from the North Atlantic Ocean, the deep Mediterranean Sea, and the Pacific Ocean oxygen minimum zone (OMZ). For this proof of concept, sixty single viruses were selected at random for sequencing. Genome annotation identified 27 of these genomes as bona fide viruses, and detected three auxiliary metabolic genes involved in nucleotide biosynthesis and sugar metabolism. Massive protein profile analysis confirmed that these viruses represented novel viral groups not present in databases. Although they were not previously assembled by viromics, global fragment recruitment analysis showed a conserved profile of relative abundance of these viruses in all analyzed samples spanning different oceans. Altogether, these results reveal the feasibility in using SVGs in this vast environment to unveil the genomes of relevant viruses.

Caudovirales bacteriophages are associated with improved executive function and memory in flies, mice, and humans.

Growing evidence implicates the gut microbiome in cognition. Viruses, the most abundant life entities on the planet, are a commonly overlooked component of the gut virome, dominated by the Caudovirales and Microviridae bacteriophages. Here, we show in a discovery (n = 114) and a validation cohort (n = 942) that subjects with increased Caudovirales and Siphoviridae levels in the gut microbiome had better performance in executive processes and verbal memory. Conversely, increased Microviridae levels were linked to a greater impairment in executive abilities. Microbiota transplantation from human donors with increased specific Caudovirales (>90% from the Siphoviridae family) levels led to increased scores in the novel object recognition test in mice and up-regulated memory-promoting immediate early genes in the prefrontal cortex. Supplementation of the Drosophila diet with the 936 group of lactococcal Siphoviridae bacteriophages resulted in increased memory scores and upregulation of memory-involved brain genes. Thus, bacteriophages warrant consideration as novel actors in the microbiome-brain axis.

Unexpected myriad of co-occurring viral strains and species in one of the most abundant and microdiverse viruses on Earth.

Viral genetic microdiversity drives adaptation, pathogenicity, and speciation and has critical consequences for the viral-host arms race occurring at the strain and species levels, which ultimately impact microbial community structure and biogeochemical cycles. Despite the fact that most efforts have focused on viral macrodiversity, little is known about the microdiversity of ecologically important viruses on Earth. Recently, single-virus genomics discovered the putatively most abundant ocean virus in temperate and tropical waters: the uncultured dsDNA virus vSAG 37-F6 infecting Pelagibacter, the most abundant marine bacteria. In this study, we report the cooccurrence of up to ≈1,500 different viral strains (>95% nucleotide identity) and ≈30 related species (80-95% nucleotide identity) in a single oceanic sample. Viral microdiversity was maintained over space and time, and most alleles were the result of synonymous mutations without any apparent adaptive benefits to cope with host translation codon bias and efficiency. Gene flow analysis used to delimitate species according to the biological species concept (BSC) revealed the impact of recombination in shaping vSAG 37-F6 virus and Pelagibacter speciation. Data demonstrated that this large viral microdiversity somehow mirrors the host species diversity since ≈50% of the 926 analyzed Pelagibacter genomes were found to belong to independent BSC species that do not significantly engage in gene flow with one another. The host range of this evolutionarily successful virus revealed that a single viral species can infect multiple Pelagibacter BSC species, indicating that this virus crosses not only formal BSC barriers but also biomes since viral ancestors are found in freshwater.

Single-virus genomics and beyond.

Viruses are extremely diverse and modulate important biological and ecological processes globally. However, much of viral diversity remains uncultured and yet to be discovered. Several powerful culture-independent tools, in particular metagenomics, have substantially advanced virus discovery. Among those tools is single-virus genomics, which yields sequenced reference genomes from individual sorted virus particles without the need for cultivation. This new method complements virus culturing and metagenomic approaches and its advantages include targeted investigation of specific virus groups and investigation of genomic microdiversity within viral populations. In this Review, we provide a brief history of single-virus genomics, outline how this emergent method has facilitated advances in virus ecology and discuss its current limitations and future potential. Finally, we address how this method may synergistically intersect with other single-virus and single-cell approaches.

Cationic Dendrimer G2-S16 Inhibits Herpes Simplex Type 2 Infection and Protects Mice Vaginal Microbiome.

The G2-S16 polyanionic carbosilane dendrimer is a promising microbicide that inhibits HSV-2 infection in vitro and in vivo in mice models. This G2-S16 dendrimer inhibits HSV-2 infection even in the presence of semen. Murine models, such as BALB/c female mice, are generally used to characterize host-pathogen interactions within the vaginal tract. However, the composition of endogenous vaginal flora remains largely undefined with modern microbiome analyses. It is important to note that the G2-S16 dendrimer does not change healthy mouse vaginal microbiome where Pseudomonas (10.2-79.1%) and Janthinobacterium (0.7-13%) are the more abundant genera. The HSV-2 vaginally infected female mice showed a significant microbiome alteration because an increase of Staphylococcus (up to 98.8%) and Escherichia (30.76%) levels were observed becoming these bacteria the predominant genera. BALB/c female mice vaginally-treated with the G2-S16 dendrimer and infected with the HSV-2 maintained a healthy vaginal microbiome similar to uninfected female mice. Summarizing, the G2-S16 polyanionic carbosilane dendrimer inhibits the HSV-2 infection in the presence of semen and prevents the alteration of mice female vaginal microbiome.

Diel cycling of the cosmopolitan abundant Pelagibacter virus 37-F6: one of the most abundant viruses on earth.

The spatiotemporal dynamics for marine viral populations has only recently been explored. However, nothing is known about temporal activities of the uncultured Pelagibacter virus vSAG 37-F6, which was discovered by single-virus genomics as potentially the most abundant marine virus. Here, we investigate the diel cycling of 37-F6 virus and the putative SAR11 host using coastal and oceanic transcriptomic and viromic time-series data from Osaka Bay and North Pacific Subtropical Gyre. Virus 37-F6 and relatives displayed diel cycling of transcriptional activities synchronized with its putative host. In both virus and host, the lowest transcription rates were observed at 14:00-15:00, coinciding roughly with maximum solar irradiance, while higher transcriptional rates were detected during the night/early morning and afternoon. Diel abundance of free viruses of 37-F6 in seawater roughly mirrored the transcriptional activities of both virus and host. In Osaka Bay, among viral relatives (genus level), virus 37-F6 specifically showed the highest ratio of transcriptional activity to virome abundance, a proxy for viral transcriptional activity relative to free viral particle abundance. This high ratio suggests high infection rate efficiencies in vSAG 37-F6 virus compared to viral relatives. Thus, time-series data revealed temporal transcript activities in one of the most abundant viruses in Earth.

Ecogenomics of the SAR11 clade.

Members of the SAR11 clade, despite their high abundance, are often poorly represented by metagenome-assembled genomes. This fact has hampered our knowledge about their ecology and genetic diversity. Here we examined 175 SAR11 genomes, including 47 new single-amplified genomes. The presence of the first genomes associated with subclade IV suggests that, in the same way as subclade V, they might be outside the proposed Pelagibacterales order. An expanded phylogenomic classification together with patterns of metagenomic recruitment at a global scale have allowed us to define new ecogenomic units of classification (genomospecies), appearing at different, and sometimes restricted, metagenomic data sets. We detected greater microdiversity across the water column at a single location than in samples collected from similar depth across the global ocean, suggesting little influence of biogeography. In addition, pangenome analysis revealed that the flexible genome was essential to shape genomospecies distribution. In one genomospecies preferentially found within the Mediterranean, a set of genes involved in phosphonate utilization was detected. While another, with a more cosmopolitan distribution, was unique in having an aerobic purine degradation pathway. Together, these results provide a glimpse of the enormous genomic diversity within this clade at a finer resolution than the currently defined clades.

Absolute quantification of infecting viral particles by chip-based digital polymerase chain reaction.

In silico and empirical quantification of viruses is paramount for obtaining information on viral populations that have a major impact on biogeochemical cycles. The uncultured Pelagibacter virus vSAG 37-F6 discovered via single-virus genomics is one of the most abundant and cosmopolitan marine viruses; however, little is understood about its temporal variation. Here, we estimated the absolute number of infecting 37-F6 viruses in coastal bacterioplankton from the Mediterranean Sea by using a novel, feasible SYBR Green I chip-based digital PCR (SYBR dPCR) technique, not implemented before for enumerating (uncultured) microbes. Quantitative SYBR dPCR estimated 450-3480 genome copies of virus 37-F6 in cells/mL (i.e. infecting viruses) and a total of ≈10-400 putative infected cells/mL with a potential C release of 0.12-4.9 pg/ml in the analysed samples. Considering that virus 37-F6 is ubiquitous and abundant in all Tara samples, an enormous amount of C could be transformed by this virus through the 'viral shunt'. Thus, this SYBR dPCR technique has enabled the absolute quantification of an ecologically relevant uncultured virus in nature and the estimation of its potential contribution on biogeochemical cycles. Overall, our study also shows that this approach has a broad applicability for quantifying any other target loci in Microbiology and Virology.

Insights into the antibiotic resistance dissemination in a wastewater effluent microbiome: bacteria, viruses and vesicles matter.

Wastewater treatment plants effluents are considered as hotspots for the dispersion of antibiotic resistance genes (ARGs) into natural ecosystems. The bacterial resistome (ARG collection in a metagenome) analyses have provided clues on antibacterial resistance dynamics. However, viruses and vesicles are frequently ignored. Here, we addressed the bacterial, viral and vesicle resistomes from a representative wastewater effluent in natural conditions and amended with polymyxin, which is used as a last resort antibiotic. Metagenomics showed that the natural prokaryotic resistome was vast (9000 ARG hits/Gb metagenome) and diverse, while viral resistome was two orders of magnitude lower (50 ARG hits/Gb metagenome) suggesting that viruses rarely encoded ARGs. After polymyxin amendment, data showed no ARG enrichment - including to polymyxin - in the microbiome. Remarkably, microbiomes responded to polymyxin with a vast release of putative vesicles (threefold increase compared with the control), which might be used as 'traps' to decrease the antibiotic concentration. Intriguingly, although polymyxin resistance genes (PRGs) were rare in the microbiome (0.018% of total ARG found), in the viral and vesicle fractions, PRGs were more abundant (0.5%-0.8% of total ARG found). Our data suggest that vesicles could have a more active role in the context of transmission of antibiotic resistances.

Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses.

Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270-14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.

Single-cell genomics uncover Pelagibacter as the putative host of the extremely abundant uncultured 37-F6 viral population in the ocean.

The identification of relevant virus-host pairs that globally account for a large pool of carbon and nutrients in the ocean is paramount to build accurate ecological models. A previous work using single-virus genomics led to the discovery of the uncultured single-virus vSAG 37-F6, originally sorted from the Mediterranean Sea (Blanes Bay Microbial Observatory), that represents one of the most abundant dsDNA viral population in the marine surface virosphere. Here, from same sampling site, we report that a Pelagibacter single-cell contained a viral member of vSAG 37-F6 population, by means of PCR screening of sorted, genome-amplified single cells with vSAG 37-F6-specific primers and whole-genome sequencing. Furthermore, viruses from this population were also found in three other Pelagibacter single cells from the South Pacific and Atlantic oceans. These new uncultured pelagiphages were genetically different from the previously characterized pelagiphage isolates. Data showed that the uncultured vSAG 37-F6 population represents the Pelagibacter phages that inhabit the sunlit ocean better, and contains a vast unrecognized microdiversity.

Deciphering the Human Virome with Single-Virus Genomics and Metagenomics.

Single-cell genomics has unveiled the metabolic potential of dominant microbes inhabiting different environments, including the human body. The lack of genomic information for predominant microbes of the human body, such as bacteriophages, hinders our ability to answer fundamental questions about our viral communities. Here, we applied single-virus genomics (SVGs) to natural human salivary samples in combination with viral metagenomics to gain some insights into the viral community structure of the oral cavity. Saliva samples were processed for viral metagenomics (n = 15) and SVGs (n = 3). A total of 1328 uncultured single viruses were sorted by fluorescence-activated virus sorting followed by whole genome amplification. Sequencing of 24 viral single amplified genomes (vSAGs) showed that half of the vSAGs contained viral hallmark genes. Among those bona fide viruses, the uncultured single virus 92-C13 putatively infecting oral Streptococcus-like species was within the top ≈10 most abundant viruses in the oral virome. Viral gene network and viral metagenomics analyses of 439 oral viruses from cultures, metagenomics, and SVGs revealed that salivary viruses were tentatively structured into ≈200 major viral clusters, corresponding to approximately genus-level groupings. Data showed that none of the publicly available viral isolates, excepting an Actinomyces phage, were significantly abundant in the oral viromes. In addition, none of the obtained viral contigs and vSAGs from this study were present in all viromes. Overall, the data demonstrates that most viral isolates are not naturally abundant in saliva, and furthermore, the predominant viruses in the oral cavity are yet uncharacterized. Results suggest a variable, complex, and interpersonal viral profile. Finally, we demonstrated the power of SVGs in combination with viral metagenomics to unveil the genetic information of the uncultured viruses of the human virome.

Single-virus genomics reveals hidden cosmopolitan and abundant viruses.

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.

Developing biological resource banks as a supporting tool for wildlife reproduction and conservation The Iberian lynx bank as a model for other endangered species.

This work presents a Biological Resource Bank generated as a complementary supporting tool for the reproduction and the in situ and ex situ conservation of the Iberian lynx. In its design we prioritized the preservation of a maximum of the current genetic and biological diversity of the population, and the harmless collection of the samples. To provide future reproductive opportunities through any possible technique, we processed and cryopreserved germinal cells and tissues from dead animals, 7 males and 6 females, as well as somatic cells and tissues from 69 different individuals. This somatic cell reserve reflects a very important fraction of the population biodiversity which, furthermore, will allow the development of a wide variety of studies that can be easily extrapolated to the majority of the population. We have developed a new non-destructive method to isolate cells with stem-cell-like properties. If considered convenient in the future, and after proper research, such cells could permit therapeutic applications and perhaps be a good source to be used in somatic cell nuclear transfer. Samples of whole blood and its derivatives, hairs, urine and feces from many different individuals were also preserved. Proper storage of such samples is required to allow epidemiological studies to be performed for the testing of different etiological hypotheses or, in general, to develop any bio-sanitary study to improve conservation strategies within the natural habitat. This work describes the main aspects involved in the practical implementation of the Iberian lynx Biological Resource Bank, as a model that could be useful for the development of similar banks for other endangered species.