Multiplexable and Scalable Aqueous Synthesis Platform for Oleate-Based, Bilayer-Coated Gold Nanoparticles.

Despite gold-based nanomaterials having a unique role in nanomedicine, among other fields, synthesis limitations relating to reaction scale-up and control result in prohibitively high gold nanoparticle costs. In this work, a new preparation procedure for lipid bilayer-coated gold nanoparticles in water is presented, using sodium oleate as reductant and capping agent. The seed-free synthesis not only allows for size precision (8-30 nm) but also remarkable particle concentration (10 mm Au). These reaction efficiencies allow for multiplexing and reaction standardization in 96-well plates using conventional thermocyclers, in addition to simple particle purification via microcentrifugation. Such a multiplexing approach also enables detailed spectroscopic investigation of the nonlinear growth process and dynamic sodium oleate/oleic acid self-assembly. In addition to scalability (at gram-level), resulting gold nanoparticles are stable at physiological pH, in common cell culture media, and are autoclavable. To demonstrate the versatility and applicability of the reported method, a robust ligand exchange with thiolated polyethylene glycol analogues is also presented.

Microbial activity contributes to spatial heterogeneity of wetland methane fluxes.

The emission of methane from wetlands is spatially heterogeneous, as concurrently measured surface fluxes can vary by orders of magnitude within the span of a few meters. Despite extensive study and the climatic significance of these emissions, it remains unclear what drives large, within-site variations. While geophysical factors (e.g., soil temperature) are known to correlate with methane (CH4) flux, measurable variance in these parameters often declines as spatial and temporal scales become finer. As methane emitted from wetlands is the direct, net product of microbial metabolisms which both produce and degrade CH4, it stands to reason that characterizing the spatial variability of microbial communities within a wetland-both horizontally and vertically-may help explain observed variances in flux. To that end, we surveyed microbial communities to a depth of 1 m across an ombrotrophic peat bog in Maine, USA using amplicon sequencing and gene expression techniques. Surface methane fluxes and geophysical factors were concurrently measured. Across the first meter of peat at the site, we observed significant changes in the abundance and composition of methanogenic taxa at every depth sampled, with variance in methanogen abundance explaining 70% of flux heterogeneity at a subset of plots. Among measured environmental factors, only peat depth emerged as correlated with flux, and had significant impact on the abundance and composition of methane-cycling communities. These conclusions suggest that a heightened awareness of how microbial communities are structured and spatially distributed within wetlands could offer improved insights into predicting CH4 flux dynamics. IMPORTANCE Globally, wetlands are one of the largest sources of methane (CH4), a greenhouse gas with a warming impact significantly greater than CO2. Methane produced in wetlands is the byproduct of a group of microorganisms which convert organic carbon into CH4. Despite our knowledge of how this process works, it is still unclear what drives dramatic, localized (<10 m) variance in emission rates from the surface of wetlands. While environmental conditions, like soil temperature or water table depth, correlate with methane flux when variance in these factors is large (e.g., spring vs fall), the explanatory power of these variables decline when spatial and temporal scales become smaller. As methane fluxes are the direct product of microbial activity, we profiled how the microbial community varied, both horizontally and vertically, across a peat bog in Maine, USA, finding that variance in microbial communities was likely contributing to much of the observed variance in flux.

Development and Application of a Polydimethylsiloxane-Based Passive Air Sampler to Assess Personal Exposure to SARS-CoV-2.

Exhaled respiratory droplets and aerosols can carry infectious viruses and are an important mode of transmission for COVID-19. Recent studies have been successful in detecting airborne SARS-CoV-2 RNA in indoor settings using active sampling methods. The cost, size, and maintenance of these samplers, however, limit their long-term monitoring ability in high-risk transmission areas. As an alternative, passive samplers can be small, lightweight, and inexpensive and do not require electrical power or maintenance for continual operation. Integration of passive samplers into wearable designs can be used to better understand personal exposure to the respiratory virus. This study evaluated the use of a polydimethylsiloxane (PDMS)-based passive sampler to assess personal exposure to aerosol and droplet SARS-CoV-2. The rate of uptake of virus-laden aerosol on PDMS was determined in lab-based rotating drum experiments to estimate time-weighted averaged airborne viral concentrations from passive sampler viral loading. The passive sampler was then embedded in a wearable clip design and distributed to community members across Connecticut to surveil personal SARS-CoV-2 exposure. The virus was detected on clips worn by five of the 62 participants (8%) with personal exposure ranging from 4 to 112 copies of SARS-CoV-2 RNA/m3, predominantly in indoor restaurant settings. Our findings demonstrate that PDMS-based passive samplers may serve as a useful exposure assessment tool for airborne viral exposure in real-world high-risk settings and provide avenues for early detection of potential cases and guidance on site-specific infection control protocols that preempt community transmission.

Development of CRISPR-Cas9 knock-in tools for free fatty acid production using the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973.

Synechococcus elongatus UTEX 2973 has one of the fastest measured doubling time of cyanobacteria making it an important candidate for metabolic engineering. Traditional genetic engineering methods, which rely on homologous recombination, however, are inefficient, labor-intensive, and time-consuming due to the oligoploidy or polyploidy nature of cyanobacteria and the reliance on unique antibiotic resistance markers. CRISPR-Cas9 has emerged as an effective and versatile editing platform in a wide variety of organisms, but its application for cyanobacterial engineering is limited by the inherent toxicity of Cas9 resulting in poor transformation efficiencies. Here, we demonstrated that a single-plasmid CRISPR-Cas9 system, pCRISPOmyces-2, can effectively knock-in a truncated thioesterase gene from Escherichia coli to generate free fatty acid (FFA) producing mutants of Syn2973. To do so, three parameters were evaluated on the effect of generating recipient colonies after conjugation with pCRISPOmyces-2-based plasmids: 1) a modified conjugation protocol termed streaked conjugation, 2) the deletion of the gene encoding RecJ exonuclease, and 3) single guide RNA (sgRNA) sequence. With the use of the streaked conjugation protocol and a ΔrecJ mutant strain of Syn2973, the conjugation efficiency for the pCRISPomyces-2 plasmid could be improved by 750-fold over the wildtype (WT) for a conjugation efficiency of 2.0 × 10-6 transconjugants/recipient cell. While deletion of the RecJ exonuclease alone increased the conjugation efficiency by 150-fold over the WT, FFA generation was impaired in FFA-producing mutants with the ΔrecJ background, and the large number of poor FFA-producing isolates indicated the potential increase in spontaneous mutation rates. The sgRNA sequence was found to be critical in achieving the desired CRISPR-Cas9-mediated knock-in mutation as the sgRNA impacts conjugation efficiency, likelihood of homogenous recombinants, and free fatty acid production in engineered strains.

Measurement of SARS-CoV-2 RNA in wastewater tracks community infection dynamics.

We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in primary sewage sludge in the New Haven, Connecticut, USA, metropolitan area during the Coronavirus Disease 2019 (COVID-19) outbreak in Spring 2020. SARS-CoV-2 RNA was detected throughout the more than 10-week study and, when adjusted for time lags, tracked the rise and fall of cases seen in SARS-CoV-2 clinical test results and local COVID-19 hospital admissions. Relative to these indicators, SARS-CoV-2 RNA concentrations in sludge were 0-2 d ahead of SARS-CoV-2 positive test results by date of specimen collection, 0-2 d ahead of the percentage of positive tests by date of specimen collection, 1-4 d ahead of local hospital admissions and 6-8 d ahead of SARS-CoV-2 positive test results by reporting date. Our data show the utility of viral RNA monitoring in municipal wastewater for SARS-CoV-2 infection surveillance at a population-wide level. In communities facing a delay between specimen collection and the reporting of test results, immediate wastewater results can provide considerable advance notice of infection dynamics.

Correction to: The chemodiversity of paddy soil dissolved organic matter correlates with microbial community at continental scales.

An amendment to this paper has been published and can be accessed via the original article.

Mice Fed an Obesogenic Western Diet, Administered Antibiotics, and Subjected to a Sterile Surgical Procedure Develop Lethal Septicemia with Multidrug-Resistant Pathobionts.

Despite antibiotics and sterile technique, postoperative infections remain a real and present danger to patients. Recent estimates suggest that 50% of the pathogens associated with postoperative infections have become resistant to the standard antibiotics used for prophylaxis. Risk factors identified in such cases include obesity and antibiotic exposure. To study the combined effect of obesity and antibiotic exposure on postoperative infection, mice were allowed to gain weight on an obesogenic Western-type diet (WD), administered antibiotics and then subjected to an otherwise recoverable sterile surgical injury (30% hepatectomy). The feeding of a WD alone resulted in a major imbalance of the cecal microbiota characterized by a decrease in diversity, loss of Bacteroidetes, a bloom in Proteobacteria, and the emergence of antibiotic-resistant organisms among the cecal microbiota. When WD-fed mice were administered antibiotics and subjected to 30% liver resection, lethal sepsis, characterized by multiple-organ damage, developed. Notable was the emergence and systemic dissemination of multidrug-resistant (MDR) pathobionts, including carbapenem-resistant, extended-spectrum ß-lactamase-producing Serratia marcescens, which expressed a virulent and immunosuppressive phenotype. Analysis of the distribution of exact sequence variants belonging to the genus Serratia suggested that these strains originated from the cecal mucosa. No mortality or MDR pathogens were observed in identically treated mice fed a standard chow diet. Taken together, these results suggest that consumption of a Western diet and exposure to certain antibiotics may predispose to life-threating postoperative infection associated with MDR organisms present among the gut microbiota.IMPORTANCE Obesity remains a prevalent and independent risk factor for life-threatening infection following major surgery. Here, we demonstrate that when mice are fed an obesogenic Western diet (WD), they become susceptible to lethal sepsis with multiple organ damage after exposure to antibiotics and an otherwise-recoverable surgical injury. Analysis of the gut microbiota in this model demonstrates that WD alone leads to loss of Bacteroidetes, a bloom of Proteobacteria, and evidence of antibiotic resistance development even before antibiotics are administered. After antibiotics and surgery, lethal sepsis with organ damage developed in in mice fed a WD with the appearance of multidrug-resistant pathogens in the liver, spleen, and blood. The importance of these findings lies in exposing how the selective pressures of diet, antibiotic exposure, and surgical injury can converge on the microbiome, resulting in lethal sepsis and organ damage without the introduction of an exogenous pathogen.

Bacterial and Archaeal Viruses of Himalayan Hot Springs at Manikaran Modulate Host Genomes.

Hot spring-associated viruses, particularly the archaeal viruses, remain under-examined compared to bacteriophages. Previous metagenomic studies of the Manikaran hot springs in India suggested an abundance of viral DNA, which prompted us to examine the virus-host (bacterial and archaeal) interactions in sediment and microbial mat samples collected from the thermal discharges. Here, we characterize the viruses (both bacterial and archaeal) from this Himalayan hot spring using both metagenomics assembly and electron microscopy. We utilized four shotgun samples from sediment (78-98°C) and two from microbial mats (50°C) to reconstruct 65 bacteriophage genomes (24-200 kb). We also identified 59 archaeal viruses that were notably abundant across the sediment samples. Whole-genome analyses of the reconstructed bacteriophage genomes revealed greater genomic conservation in sediments (65%) compared to microbial mats (49%). However, a minimal phage genome was still maintained across both sediment and microbial mats suggesting a common origin. To complement the metagenomic data, scanning-electron and helium-ion microscopy were used to reveal diverse morphotypes of Caudovirales and archaeal viruses. The genome level annotations provide further evidence for gene-level exchange between virus and host in these hot springs, and augments our knowledgebase for bacteriophages, archaeal viruses and Clustered Regularly Interspaced Short Palindromic Repeat cassettes, which provide a critical resource for studying viromes in extreme natural environments.

The chemodiversity of paddy soil dissolved organic matter correlates with microbial community at continental scales.

BACKGROUND: Paddy soil dissolved organic matter (DOM) represents a major hotspot for soil biogeochemistry, yet we know little about its chemodiversity let alone the microbial community that shapes it. Here, we leveraged ultrahigh-resolution mass spectrometry, amplicon, and metagenomic sequencing to characterize the molecular distribution of DOM and the taxonomic and functional microbial diversity in paddy soils across China. We hypothesized that variances in microbial community significantly associate with changes in soil DOM molecular composition. RESULTS: We report that both microbial and DOM profiles revealed geographic patterns that were associated with variation in mean monthly precipitation, mean annual temperature, and pH. DOM molecular diversity was significantly correlated with microbial taxonomic diversity. An increase in DOM molecules categorized as peptides, carbohydrates, and unsaturated aliphatics, and a decrease in those belonging to polyphenolics and polycyclic aromatics, significantly correlated with proportional changes in some of the microbial taxa, such as Syntrophobacterales, Thermoleophilia, Geobacter, Spirochaeta, Gaiella, and Defluviicoccus. DOM composition was also associated with the relative abundances of the microbial metabolic pathways, such as anaerobic carbon fixation, glycolysis, lignolysis, fermentation, and methanogenesis. CONCLUSIONS: Our study demonstrates the continental-scale distribution of DOM is significantly correlated with the taxonomic profile and metabolic potential of the rice paddy microbiome. Abiotic factors that have a distinct effect on community structure can also influence the chemodiversity of DOM and vice versa. Deciphering these associations and the underlying mechanisms can precipitate understanding of the complex ecology of paddy soils, as well as help assess the effects of human activities on biogeochemistry and greenhouse gas emissions in paddy soils.