Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

Efforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgoniansEunicea flexuosaandGorgonia ventalina,and the scleractinianPorites panamensis. The 20-bp PNA clamp was designed using DNA fromE. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads inE. flexuosasamples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction ofE. flexuosaDNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian,G. ventalina,by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral,P. panamensis,by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities. Keyword

In Vitro Models for Studying Respiratory Host-Pathogen Interactions.

Respiratory diseases and lower respiratory tract infections are among the leading cause of death worldwide and, especially given the recent severe acute respiratory syndrome coronavirus-2 pandemic, are of high and prevalent socio-economic importance. In vitro models, which accurately represent the lung microenvironment, are of increasing significance given the ethical concerns around animal work and the lack of translation to human disease, as well as the lengthy time to market and the attrition rates associated with clinical trials. This review gives an overview of the biological and immunological components involved in regulating the respiratory epithelium system in health, disease, and infection. The evolution from 2D to 3D cell biology and to more advanced technological integrated models for studying respiratory host-pathogen interactions are reviewed and provide a reference point for understanding the in vitro modeling requirements. Finally, the current limitations and future perspectives for advancing this field are presented

Building Scaffolds for Tubular Tissue Engineering.

Hollow organs and tissue systems drive various functions in the body. Many of these hollow or tubular systems, such as vasculature, the intestines, and the trachea, are common targets for tissue engineering, given their relevance to numerous diseases and body functions. As the field of tissue engineering has developed, numerous benchtop models have been produced as platforms for basic science and drug testing. Production of tubular scaffolds for different tissue engineering applications possesses many commonalities, such as the necessity for producing an intact tubular opening and for formation of semi-permeable epithelia or endothelia. As such, the field has converged on a series of manufacturing techniques for producing these structures. In this review, we discuss some of the most common tissue engineered applications within the context of tubular tissues and the methods by which these structures can be produced. We provide an overview of the general structure and anatomy for these tissue systems along with a series of general design criteria for tubular tissue engineering. We categorize methods for manufacturing tubular scaffolds as follows: casting, electrospinning, rolling, 3D printing, and decellularization. We discuss state-of-the-art models within the context of vascular, intestinal, and tracheal tissue engineering. Finally, we conclude with a discussion of the future for these fields

Identifying the plant‐associated microbiome across aquatic and terrestrial environments: the effects of amplification method on taxa discovery

Plants in terrestrial and aquatic environments contain a diverse microbiome. Yet, the chloroplast and mitochondria organelles of the plant eukaryotic cell originate from free‐living cyanobacteria and Rickettsiales. This represents a challenge for sequencing the plant microbiome with universal primers, as ~99% of 16S rRNA sequences may consist of chloroplast and mitochondrial sequences. Peptide nucleic acid clamps offer a potential solution by blocking amplification of host‐associated sequences. We assessed the efficacy of chloroplast and mitochondria‐blocking clamps against a range of microbial taxa from soil, freshwater and marine environments. While we found that the mitochondrial blocking clamps appear to be a robust method for assessing animal‐associated microbiota, Proteobacterial 16S rRNA binds to the chloroplast‐blocking clamp, resulting in a strong sequencing bias against this group. We attribute this bias to a conserved 14‐bp sequence in the Proteobacteria that matches the 17‐bp chloroplast‐blocking clamp sequence. By scanning the Greengenes database, we provide a reference list of nearly 1500 taxa that contain this 14‐bp sequence, including 48 families such as the Rhodobacteraceae, Phyllobacteriaceae, Rhizobiaceae, Kiloniellaceae and Caulobacteraceae. To determine where these taxa are found in nature, we mapped this taxa reference list against the Earth Microbiome Project database. These taxa are abundant in a variety of environments, particularly aquatic and semiaquatic freshwater and marine habitats. To facilitate informed decisions on effective use of organelle‐blocking clamps, we provide a searchable database of microbial taxa in the Greengenes and Silva databases matching various n‐mer oligonucleotides of each PNA sequence.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138887/1/men12645.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138887/2/men12645_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138887/3/men12645-sup-0001-SupInfo.pd

Restricted streptomycin use in apple orchards did not adversely alter the soil bacteria communities

Streptomycin has been authorized for restricted use in the prevention of the fire blight disease of pome fruit orchards in the EU and Switzerland. This study addresses the important topic of the influence of the use of streptomycin in agriculture on the total bacteria community within the soil ecosystem. Soil samples were taken from soils under apple trees, prior to streptomycin application and 2 weeks post streptomycin application or water application (untreated control). High throughput 16S rRNA gene amplicon sequencing was used to generate datasets from the soils under apple trees in apple orchards from three different locations in Switzerland. We hypothesized that the use of streptomycin would reduce the bacterial diversity within the soil samples and enhance a reduction in the variety of taxa present. Bacterial species such as Pseudomonas, Burkholderia, and Stenotrophomonas are intrinsically resistant to many antibiotics and as such it is of interest to investigate if the use of streptomycin provided a selective advantage for these bacteria in the soil ecosystem. The application of streptomycin did not influence the abundance and diversities of major bacteria taxa of the soils or the Pseudomonas, Burkholderia, and Stenotrophomonas species. We also discovered that apple orchards under the same management practices, did not harbor the same bacterial communities. The restricted application of streptomycin in the protection of apple orchards from the fire blight pathogen Erwinia amylovora under the guidelines in Switzerland did not alter either the bacterial diversity or abundance within these soil ecosystems

Acute Arterial Thrombosis after Covered Stent Exclusion of Bleeding Mycotic Pseudoaneurysm: Treatment Using Catheter-Directed Thrombolysis

Conventional absolute contraindications to catheter-directed thrombolysis include active or recent hemorrhage and the presence of local vascular infection, both of which increase the risk of procedure-related complications such as bleeding and systemic sepsis. For this reason, lytic therapy of arterial thromboembolism under these circumstances is generally precluded. Herein, we describe a unique case of safe catheter-directed lysis of an acutely thrombosed iliac artery following covered stent placement for treatment of an actively bleeding infected pseudoaneurysm. Our management approach is discussed

Saliva from obese individuals suppresses the release of aroma compounds from wine.

BackgroundRecent evidence suggests that a lower extent of the retronasal aroma release correspond to a higher amount of ad libitum food intake. This has been regarded as one of the bases of behavioral choices towards food consumption in obese people. In this pilot study we investigated the hypothesis that saliva from obese individuals could be responsible for an alteration of the retro-nasal aroma release. We tested this hypothesis in vitro, by comparing the release of volatiles from a liquid food matrix (wine) after its interaction with saliva from 28 obese (O) and 28 normal-weight (N) individuals.Methods and findingsAmplicon sequencing of the 16S rRNA V4 region indicated that Firmicutes and Actinobacteria were more abundant in O, while Proteobacteria and Fusobacteria dominated in N. Streptococcaceae were significantly more abundant in the O subjects and constituted 34% and 19% on average of the saliva microbiota of O and N subjects, respectively. The Total Antioxidant Capacity was higher in O vs N saliva samples. A model mouth system was used to test whether the in-mouth wine aroma release differs after the interaction with O or N saliva. In O samples, a 18% to 60% significant decrease in the mean concentration of wine volatiles was detected as a result of interaction with saliva, compared with N. This suppression was linked to biochemical differences in O and N saliva composition, which include protein content.ConclusionMicrobiological and biochemical differences were found in O vs N saliva samples. An impaired retronasal aroma release from white wine was detected in vitro and linked to compositional differences between saliva from obese and normal-weight subjects. Additional in vivo investigations on diverse food matrices could contribute to understanding whether a lower olfactory stimulation due to saliva composition can be a co-factor in the development/maintenance of obesity

Investigating the Impact of Storage Conditions on Microbial Community Composition in Soil Samples

Recent advances in DNA sequencing technologies have allowed scientists to probe increasingly complex biological systems, including the diversity of bacteria in the environment. However, despite a multitude of recent studies incorporating these methods, many questions regarding how environmental samples should be collected and stored still persist. Here, we assess the impact of different soil storage conditions on microbial community composition using Illumina-based 16S rRNA V4 amplicon sequencing. Both storage time and temperature affected bacterial community composition and structure. Frozen samples maintained the highest alpha diversity and differed least in beta diversity, suggesting the utility of cold storage for maintaining consistent communities. Samples stored for intermediate times (three and seven days) had both the highest alpha diversity and the largest differences in overall beta diversity, showing the degree of community change after sample collection. These divergences notwithstanding, differences in neither storage time nor storage temperature substantially altered overall communities relative to more than 500 previously examined soil samples. These results systematically support previous studies and stress the importance of methodological consistency for accurate characterization and comparison of soil microbiological assemblages.</p

DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied