An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing.

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.

Impact of Beer and Nonalcoholic Beer Consumption on the Gut Microbiota: A Randomized, Double-Blind, Controlled Trial.

Gut microbiota modulation might constitute a mechanism mediating the effects of beer on health. In this randomized, double-blinded, two-arm parallel trial, 22 healthy men were recruited to drink 330 mL of nonalcoholic beer (0.0% v/v) or alcoholic beer (5.2% v/v) daily during a 4-week follow-up period. Blood and faecal samples were collected before and after the intervention period. Gut microbiota was analyzed by 16S rRNA gene sequencing. Drinking nonalcoholic or alcoholic beer daily for 4 weeks did not increase body weight and body fat mass and did not changed significantly serum cardiometabolic biomarkers. Nonalcoholic and alcoholic beer increased gut microbiota diversity which has been associated with positive health outcomes and tended to increase faecal alkaline phosphatase activity, a marker of intestinal barrier function. These results suggest the effects of beer on gut microbiota modulation are independent of alcohol and may be mediated by beer polyphenols.

Gut Microbiota, in the Halfway between Nutrition and Lung Function.

The gut microbiota is often mentioned as a "forgotten organ" or "metabolic organ", given its profound impact on host physiology, metabolism, immune function and nutrition. A healthy diet is undoubtedly a major contributor for promoting a "good" microbial community that turns out to be crucial for a fine-tuned symbiotic relationship with the host. Both microbial-derived components and produced metabolites elicit the activation of downstream cascades capable to modulate both local and systemic immune responses. A balance between host and gut microbiota is crucial to keep a healthy intestinal barrier and an optimal immune homeostasis, thus contributing to prevent disease occurrence. How dietary habits can impact gut microbiota and, ultimately, host immunity in health and disease has been the subject of intense study, especially with regard to metabolic diseases. Only recently, these links have started to be explored in relation to lung diseases. The objective of this review is to address the current knowledge on how diet affects gut microbiota and how it acts on lung function. As the immune system seems to be the key player in the cross-talk between diet, gut microbiota and the lungs, involved immune interactions are discussed. There are key nutrients that, when present in our diet, help in gut homeostasis and lead to a healthier lifestyle, even ameliorating chronic diseases. Thus, with this review we hope to incite the scientific community interest to use diet as a valuable non-pharmacological addition to lung diseases management. First, we talk about the intestinal microbiota and interactions through the intestinal barrier for a better understanding of the following sections, which are the main focus of this article: the way diet impacts the intestinal microbiota and the immune interactions of the gut-lung axis that can explain the impact of diet, a key modifiable factor influencing the gut microbiota in several lung diseases.

Natural hot spots for gain of multiple resistances: arsenic and antibiotic resistances in heterotrophic, aerobic bacteria from marine hydrothermal vent fields.

Microorganisms are responsible for multiple antibiotic resistances that have been associated with resistance/tolerance to heavy metals, with consequences to public health. Many genes conferring these resistances are located on mobile genetic elements, easily exchanged among phylogenetically distant bacteria. The objective of the present work was to isolate arsenic-, antimonite-, and antibiotic-resistant strains and to determine the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in phenotypically resistant strains, in a nonanthropogenically impacted environment. The hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between 11°N and 38°N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention, was sampled as a metal-rich pristine environment. A total of 35 strains from 8 different species were isolated in the presence of arsenate, arsenite, and antimonite. ACR3 and arsB genes were amplified from the sediment's total DNA, and 4 isolates also carried ACR3 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had recoverable plasmids. Purified plasmids were sequenced by Illumina and assembled by EDENA V3, and contig annotation was performed using the "Rapid Annotation using the Subsystems Technology" server. Determinants of resistance to copper, zinc, cadmium, cobalt, and chromium as well as to the antibiotics ß-lactams and fluoroquinolones were found in the 3 sequenced plasmids. Genes coding for heavy metal resistance and antibiotic resistance in the same mobile element were found, suggesting the possibility of horizontal gene transfer and distribution of theses resistances in the bacterial population.

Inactivation of bacterial and viral biothreat agents on metallic copper surfaces.

In recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. Microbes are rapidly inactivated by a quick, sharp shock known as contact killing. The underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. Pathogenic bacterial and viral high-consequence species able to evade the host immune system are among the most serious lethal microbial challenges to human health. Here, we investigated contact-killing mediated by copper surfaces of Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis tularensis and Yersinia pestis) and of Gram-positive endospore-forming Bacillus anthracis. Additionally, we also tested inactivation of monkeypox virus and vaccinia virus on copper. This group of pathogens comprises biothreat species (or their close relatives) classified by the Center for Disease and Control and Prevention (CDC) as microbial select agents posing severe threats to public health and having the potential to be deliberately released. All agents were rapidly inactivated on copper between 30 s and 5 min with the exception of B. anthracis endospores. For vegetative bacterial cells prolonged contact to metallic copper resulted in the destruction of cell structure.

Draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user.

We report the draft genome sequence of Bacillus anthracis UR-1, isolated from a fatal case of injectional anthrax in a German heroin user. Analysis of the genome sequence of strain UR-1 may aid in describing phylogenetic relationships between virulent heroin-associated isolates of B. anthracis isolated in the United Kingdom, Germany, and other European countries.

Draft genome sequence of Pseudomonas sp. strain M47T1, carried by Bursaphelenchus xylophilus isolated from Pinus pinaster.

The draft genome sequence of Pseudomonas sp. strain M47T1, carried by the Bursaphelenchus xylophilus pinewood nematode, the causative agent of pine wilt disease, is presented. In Pseudomonas sp. strain M47T1, genes that make this a plant growth-promoting bacterium, as well as genes potentially involved in nematotoxicity, were identified.

Draft genome sequence of Serratia sp. strain M24T3, isolated from pinewood disease nematode Bursaphelenchus xylophilus.

Here we report the draft genome sequence of Serratia sp. strain M24T3, which is associated with pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. Serratia sp. strain M24T3 has been identified as a bionematocide for B. xylophilus in vitro, and multiple genes potentially involved in virulence and nematotoxity were identified.

Quantitative proteomic profiling of the Escherichia coli response to metallic copper surfaces.

Metallic copper surfaces have strong antimicrobial properties and kill bacteria, such as Escherichia coli, within minutes in a process called contact killing. These bacteria are exposed to acute copper stress under dry conditions which is different from chronic copper stress in growing liquid cultures. Currently, the physiological changes of E. coli during the acute contact killing process are largely unknown. Here, a label-free, quantitative proteomic approach was employed to identify the differential proteome profiles of E. coli cells after sub-lethal and lethal exposure to dry metallic copper. Of the 509 proteins identified, 110 proteins were differentially expressed after sub-lethal exposure, whereas 136 proteins had significant differences in their abundance levels after lethal exposure to copper compared to unexposed cells. A total of 210 proteins were identified only in copper-responsive proteomes. Copper surface stress coincided with increased abundance of proteins involved in secondary metabolite biosynthesis, transport and catabolism, including efflux proteins and multidrug resistance proteins. Proteins involved in translation, ribosomal structure and biogenesis functions were down-regulated after contact to metallic copper. The set of changes invoked by copper surface-exposure was diverse without a clear connection to copper ion stress but was different from that caused by exposure to stainless steel. Oxidative posttranslational modifications of proteins were observed in cells exposed to copper but also from stainless steel surfaces. However, proteins from copper stressed cells exhibited a higher degree of oxidative proline and threonine modifications.

Mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces.

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC(2)(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.

Bacterial killing by dry metallic copper surfaces.

Metallic copper surfaces rapidly and efficiently kill bacteria. Cells exposed to copper surfaces accumulated large amounts of copper ions, and this copper uptake was faster from dry copper than from moist copper. Cells suffered extensive membrane damage within minutes of exposure to dry copper. Further, cells removed from copper showed loss of cell integrity. Acute contact with metallic copper surfaces did not result in increased mutation rates or DNA lesions. These findings are important first steps for revealing the molecular sensitive targets in cells lethally challenged by exposure to copper surfaces and provide a scientific explanation for the use of copper surfaces as antimicrobial agents for supporting public hygiene.

Roseomonas pecuniae sp. nov., isolated from the surface of a copper-alloy coin.

Strain N75(T) was isolated from the surface of a copper-alloy 50 Euro cent coin collected from general circulation. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain N75(T) formed a distinct branch within the genus Roseomonas and placed it in the Alphaproteobacteria. Strain N75(T) showed 16S rRNA gene sequence similarities of 92.4-97.1  % to type strains of species of the genus Roseomonas. Strain N75(T) was a Gram-negative, non-spore-forming, non-motile coccoid, with an optimum growth temperature of about 30 °C; the strain did not grow at 5 or 37 °C. Strain N75(T) did not grow in medium containing NaCl. The major respiratory quinone was ubiquinone 10 (Q-10). The major fatty acids were unsaturated C16:1ω7c/C16:1ω6c and C18:1ω7c (around 70 % of the total fatty acids); the third most abundant fatty acid was the hydroxylated C18:1 2-OH. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content was 72.8 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, we conclude that strain N75(T) represents a novel species of the genus Roseomonas, for which we propose the name Roseomonas pecuniae sp. nov. (type strain N75(T) =LMG 25481(T) =CIP 110074(T)).

Contribution of copper ion resistance to survival of Escherichia coli on metallic copper surfaces.

Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.