Impact of sample preservation and manipulation on insect gut microbiome profiling : a test case with fruit flies (Diptera, Tephritidae)

High-throughput sequencing (HTS) techniques are of great value for the investigation of microbial communities, and have been extensively used to study the gut microbiome. While most studies focus on the human gut, many others have investigated insects. However, because of the rapid spread of HTS techniques, a lot of variation exists in the protocols for sample preparation. In the present study, we investigated the impact of two widely adopted sample-processing procedures preceding library preparation, i.e., preservation of insect tissue in 70% ethanol (EtOH) and sample dissection. We used the fruit fly Ceratitis capitata (Diptera: Tephritidae) as a model organism and set up two experiments, one comparing the effects of sample manipulation and preservation across life stages and the other across fruit samples from different sources. The results of this study showed no major effects of dissection on the outcome of HTS. However, EtOH preservation did have effects on the recovered gut microbiome, the main effect being a significant reduction of the dominant genus, Providencia, in EtOH-preserved samples. Less abundant bacterial groups were also affected resulting in altered microbial profiles obtained from samples preserved in 70% EtOH. These results have important implications for the planning of future studies and when comparing studies that used different sample preparation protocols

Burkholderia anthina sp. nov. and Burkholderia pyrrocinia , two additional Burkholderia cepacia complex bacteria, may confound results of new molecular diagnostic tools

Nineteen Burkholderia cepacia -like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B. cepacia complex bacteria. Various genotypic and phenotypic characteristics were examined. The results of this polyphasic study allowed classification of the 19 isolates as an eighth B. cepacia complex genomovar ( Burkholderia anthina sp. nov.) and to design tools for its identification in the diagnostic laboratory. In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B. cepacia complex. This highlights another potential source for diagnostic problems with B. cepacia -like bacteria.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71564/1/j.1574-695X.2002.tb00584.x.pd

The type and concentration of inoculum and substrate as well as the presence of oxygen impact the water kefir fermentation process

Eleven series of water kefir fermentation processes differing in the presence of oxygen and the type and concentration of inoculum and substrate, were followed as a function of time to quantify the impact of these parameters on the kinetics of this process via a modeling approach. Increasing concentrations of the water kefir grain inoculum increased the water kefir fermentation rate, so that the metabolic activity during water kefir fermentation was mainly associated with the grains. Water kefir liquor could also be used as an alternative means of inoculation, but the resulting fermentation process progressed slower than the one inoculated with water kefir grains, and the production of water kefir grain mass was absent. Substitution of sucrose with glucose and/or fructose reduced the water kefir grain growth, whereby glucose was fermented faster than fructose. Lacticaseibacillus paracasei (formerly known as Lactobacillus paracasei), Lentilactobacillus hilgardii (formerly known as Lactobacillus hilgardii), Liquorilactobacillus nagelii (formerly known as Lactobacillus nagelii), Saccharomyces cerevisiae, and Dekkera bruxellensis were the main microorganisms present. Acetic acid bacteria were present in low abundances under anaerobic conditions and only proliferated under aerobic conditions. Visualization of the water kefir grains through scanning electron microscopy revealed that the majority of the microorganisms was attached onto their surface. Lactic acid bacteria and yeasts were predominantly associated with the grains, whereas acetic acid bacteria were predominantly associated with the liquor

Učinkovitost natrijevih soli hidroksiflavona kao hvatača slobodnih radikala u usporedbi s aktivnošću samih hidroksiflavona

Different studies have demonstrated that the bioactivity of hydroxyflavones is due to their antioxidant and free radical scavenging activity. Recently, most interest has been devoted to structure- activity relationships; however, the main problems encountered in these studies are the low solubility of some hydroxyflavones in aqueous solution and the pro-oxidant character of the hydroxyl group at the 3 position. In the present investigation, these problems have been resolved by preparing the corresponding hydroxyflavone sodium salts. In this way, the hydroxyl free radical (OH)• scavenging activity of different hydroxyflavone salts were studied using the Fenton reaction model, and the superoxide (O2)• – scavenging activity was studied using the xanthine oxidase model. The results obtained show clearly that the (OH)• scavenging activity of the hydroxyflavone salt form is at least two times that of the corresponding hydroxyflavone itself. Considering the (O2)• – scavenging activity, the salt form of hydroxyflavone is as good as the corresponding hydroxyflavone. Moreover, it was observed that for a good scavenging activity the hydroxyl at the 3\u27 position must be free, and only the hydroxyl groups of 3 and 4\u27 have to be substituted by sodium when the sodium salt of hydroxyflavone at position 7 does not have an important role in radical scavenging. The salt forms of hydroxyflavones are interesting free radical scavenger compounds showing a hydrophilic character.Različita istraživanja pokazala su da je bioaktivnost hidroksiflavona posljedica njihove antioksidacijske aktivnosti i sposobnosti hvatanja slobodnih radikala. Do sada je mnogo zanimanja iskazano za odnos između strukture i aktivnosti; međutim, osnovni problemi na koje se naišlo u tim istraživanjima slaba je topljivost nekih hidroksiflavona u vodenoj otopini i prooksidacijski karakter hidroksilne skupine u položaju 3. U ovom istraživanju ti problemi razriješeni su pripremom odgovarajućih natrijevih soli hidroksiflavona. Tako su proučavani učinkovitost soli raznih hidroksiflavona kao hvatača slobodnih hidroksilnih radikala (OH)• modelom Fentonove reakcije te kao hvatača superoksid radikala (O2)• – modelom ksantin oksidaze. Dobiveni rezultati jasno pokazuju da soli hidroksiflavona najmanje dva puta učinkovitije hvataju hidroksilne radikale (OH)• nego što to čine odgovarajući hidroksiflavoni. Učinkovitost hvatanja superoksid radikala (O2)• – jednaka je kako za soli hidroksiflavona tako i za same hidroksiflavone. Osim toga opaženo je da za dobar učinak hvatanja radikala, OH skupina u položaju 3\u27 treba biti slobodna, da samo hidroksilne skupine 3 i 4\u27 trebaju biti supstituirane natrijem, dok natrijeva sol hidroksiflavona u položaju 7 nema znatnijega utjecaja na hvatanje radikala. Soli hidroksiflavona, spojevi koji pokazuju hidrofilni karakter, zanimljivi su hvatači slobodnih radikala

Cell-Free DNA From Metastatic Pancreatic Neuroendocrine Tumor Patients Contains Tumor-Specific Mutations and Copy Number Variations

Background: Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. So far, use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients.Methods: From 10 PNET patients, fresh frozen tumor tissue, buffy coat and plasma samples were collected. Whole-exome sequencing of primary tumor and germline DNA was performed to identify tumor-specific variants and copy number variations (CNVs). Subsequently, tumor-specific variants were quantified in plasma cfDNA with droplet digital PCR. In addition, CNV analysis of cfDNA was performed using shallow whole-genome sequencing.Results: Tumor-specific variants were detected in perioperative plasma samples of two PNET patients, at variant allele fractions (VAFs) of respectively 19 and 21%. Both patients had metastatic disease at time of surgery, while the other patients presented with localized disease. In the metastatic patients, CNV profiles of tumor tissue and cfDNA were significantly correlated. A follow-up plasma sample of a metastatic patient demonstrated an increased VAF (57%) and an increased chromosomal instability, in parallel with an increase in tumor burden.Conclusions: We are the first to report the presence of tumor-specific genetic alterations in cfDNA of metastatic PNET patients and their evolution during disease progression. Additionally, CNV analysis in cfDNA shows potential as a liquid biopsy

Relating increasing hantavirus incidences to the changing climate: the mast connection

<p>Abstract</p> <p>Background</p> <p>Nephropathia epidemica (NE), an emerging rodent-borne viral disease, has become the most important cause of infectious acute renal failure in Belgium, with sharp increases in incidence occurring for more than a decade. Bank voles are the rodent reservoir of the responsible hantavirus and are known to display cyclic population peaks. We tried to relate these peaks to the cyclic NE outbreaks observed since 1993. Our hypothesis was that the ecological causal connection was the staple food source for voles, being seeds of deciduous broad-leaf trees, commonly called "mast". We also examined whether past temperature and precipitation preceding "mast years" were statistically linked to these NE outbreaks.</p> <p>Results</p> <p>Since 1993, each NE peak is immediately preceded by a mast year, resulting in significantly higher NE case numbers during these peaks (Spearman R = -0.82; P = 0.034). NE peaks are significantly related to warmer autumns the year before (R = 0.51; P < 0.001), hotter summers two years before (R = 0.32; P < 0.001), but also to colder (R = -0.25; P < 0.01) and more moist summers (R = 0.39; P < 0.001) three years before. Summer correlations were even more pronounced, when only July was singled out as the most representative summer month.</p> <p>Conclusion</p> <p>NE peaks in year 0 are induced by abundant mast formation in year-1, facilitating bank vole survival during winter, thus putting the local human population at risk from the spring onwards of year 0. This bank vole survival is further promoted by higher autumn temperatures in year-1, whereas mast formation itself is primed by higher summer temperatures in year-2. Both summer and autumn temperatures have been rising to significantly higher levels during recent years, explaining the virtually continuous epidemic state since 2005 of a zoonosis, considered rare until recently. Moreover, in 2007 a NE peak and an abundant mast formation occurred for the first time within the same year, thus forecasting yet another record NE incidence for 2008. We therefore predict that with the anticipated climate changes due to global warming, NE might become a highly endemic disease in Belgium and surrounding countries.</p

Assessing transmissibility of HIV-1 drug resistance mutations from treated and from drug-naive individuals (AIDS (2015) 29 (2045-2052))

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CHARACTERIZATION OF F107 FIMBRIAE OF ESCHERICHIA-COLI 107/86, WHICH CAUSES EDEMA DISEASE IN PIGS, AND NUCLEOTIDE-SEQUENCE OF THE F107 MAJOR FIMBRIAL SUBUNIT GENE, FEDA

F107 fimbriae were isolated and purified from edema disease strain 107/86 of Escherichia coli. Plasmid pIH120 was constructed, which contains the gene cluster that codes for adhesive F107 fimbriae. The major fimbrial subunit gene, fedA, was sequenced. An open reading frame that codes for a protein with 170 amino acids, including a 21-amino-acid signal peptide, was found. The protein without the signal sequence has a calculated molecular mass of 15,099 Da. Construction of a nonsense mutation in the open reading frame of fedA abolished both fimbrial expression and the capacity to adhere to isolated porcine intestinal villi. In a screening of 28 reference edema disease strains and isolates from clinically ill piglets, fedA was detected in 24 cases (85.7%). In 20 (83.3%) of these 24 strains, fedA was found in association with Shiga-like toxin II variant genes, coding for the toxin that is characteristic for edema disease strains of E. coli. The fimbrial subunit gene was not detected in enterotoxigenic E. coli strains. Because of the capacity of E. coli HB101(pIH120) transformants to adhere to isolated porcine intestinal villi, the high prevalence of fedA in edema disease strains, and the high correlation with the Shiga-like toxin II variant toxin-encoding genes, we suggest that F107 fimbriae are an important virulence factor in edema disease strains of E. coli

Enterococcus devriesei sp. nov., associated with animal sources

http://ijs.sgmjournals.org/The taxonomic position of two bovine strains, LMG 13603 and LMG 14595, assigned to the species Enterococcus raffinosus on the basis of biochemical features, was reinvestigated. Both reference strains and two other isolates, 6/1 (=LMG 22829) originating from a charcoal-broiled river lamprey and IE38.4 (=LMG 22830) from the air of a poultry slaughter by-product processing plant, occupied a clearly separate position, on the basis of sequence analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase a-subunit), relative to the type strain of E. raffinosus and all other enterococcal species with validly published names. 16S rRNA gene sequencing of strains LMG 13603, LMG 14595, 6/1 and IE38.4 confirmed their phylogenetic position in the Enterococcus avium species group, there being more than 99% 16S rRNA gene sequence similarity to most members of the group, including E. raffinosus, and revealed Enterococcus pseudoavium as the closest phylogenetic relative (99,8–99,9 %). Further phenotypic and genotypic analyses using whole-cell-protein electrophoresis, (GTG)5-PCR fingerprinting, ribotyping and DNA–DNA hybridization experiments demonstrated that all four strains represent a novel enterococcal species, for which the name Enterococcus devriesei sp. nov. is proposed. The type strain is LMG 14595T (=CCM 7299T)