MIWI N-terminal arginines orchestrate generation of functional pachytene piRNAs and spermiogenesis.

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.

MIWI arginines orchestrate generation of functional pachytene piRNAs and spermiogenesis.

N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. Surprisingly, the loss of TDRD5 and TDRKH interaction with MIWI results in defective piRNA amplification, rather than an expected failure of piRNA biogenesis. We find that piRNA amplification is necessary for both transposon control and for sustaining levels of select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as autonomous genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.

Exploring the mobilome and resistome of Enterococcus faecium in a One Health context across two continents.

Enterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, virulence factors and MGEs were profiled in a set of 1273 E. faecium genomes from two disparate geographic locations (in the UK and Canada) from a range of agricultural, clinical and associated habitats. Neither lineages of E. faecium, type A and B, nor MGEs are constrained by geographic proximity, but our results show evidence of a strong association of many profiled genes and MGEs with habitat. Many features were associated with a group of clinical and municipal wastewater genomes that are likely forming a new human-associated ecotype within type A. The evolutionary dynamics of E. faecium make it a highly versatile emerging pathogen, and its ability to acquire, transmit and lose features presents a high risk for the emergence of new pathogenic variants and novel resistance combinations. This study provides a workflow for MGE-centric surveillance of AMR in Enterococcus that can be adapted to other pathogens.

Thermal Death Kinetics of Three Representative Salmonella enterica Strains in Toasted Oats Cereal.

Several reports have indicated that the thermal tolerance of Salmonella at low-water activity increases significantly, but information on the impact of diverse food matrices is still scarce. The goal of this research was to determine the kinetic parameters (decimal reduction time, D; time required for the first decimal reduction, δ) of thermal resistance of Salmonella in a previously cooked low water activity food. Commercial toasted oats cereal (TOC) was used as the food model, with or without sucrose (25%) addition. TOC samples were inoculated with 108 CFU/mL of a single strain of one of three Salmonella serovars (Agona, Tennessee, Typhimurium). TOC samples were ground and equilibrated to aw values of 0.11, 0.33 and 0.53, respectively. Ground TOC was heated at temperatures between 65 °C and 105 °C and viable counts were determined over time (depending on the temperature for up to 6 h). Death kinetic parameters were determined using linear and Weibull regression models. More than 70% of Weibull's adjusted regression coefficients (Radj2) and only 38% of the linear model's Radj2 had values greater than 0.8. For all serovars, both D and δ values increased consistently at a 0.11 aw compared to 0.33 and 0.53. At 0.33 aw, the δ values for Typhimurium, Tennessee and Agona were 0.55, 1.01 and 2.87, respectively, at 85 °C, but these values increased to 65, 105 and 64 min, respectively, at 0.11 aw. At 100 °C, δ values were 0.9, 5.5 and 2.3 min, respectively, at 0.11 aw. The addition of sucrose resulted in a consistent reduction of eight out of nine δ values determined at 0.11 aw at 85, 95 and 100 °C, but this trend was not consistent at 0.33 and 0.53 aw. The Z values (increase of temperature required to decrease δ-value one log) were determined with modified δ values for a fixed ß (a fitting parameter that describes the shape of the curve), and ranged between 8.9 °C and 13.4 °C; they were not influenced by aw, strain or sugar content. These findings indicated that in TOC, high thermal tolerance was consistent among serovars and thermal tolerance was inversely dependent on aw.

Evaluation of the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) system in the detection of mastitis pathogens from bovine milk samples.

MALDI-TOF is a chemistry analytical tool that has recently been deployed in the identification of microorganisms isolated from nosocomial environments. Its use in diagnostics has been extremely advantageous in terms of cost effectiveness, sample preparation easiness, turn-around time and result analysis accessibility. In the dairy industry, where mastitis causes great financial losses, a rapid diagnostic method such as MALDI-TOF could assist in the control and prevention program of mastitis, in addition to the sanitation and safety level of the dairy farms and processing facility. However, the diagnostic strengths and limitations of this test method require further understanding. In the present study, we prospectively compared MALDI-TOF MS to conventional 16S rDNA sequencing method for the identification of pathogens recovered from milk associated with clinical and subclinical bovine mastitis cases. Initially, 810 bacterial isolates were collected from raw milk samples over a period of three months. However, only the isolates (481) having both 16S rDNA sequencing and MALDI-TOF identification were included in the final phase of the study. Among the 481 milk isolates, a total of 26 genera (12 g-postive and 14 g-negative), including 71 different species, were taxonomically charecterized by 16S rDNA at the species level. Comparatively, MALDI-TOF identified 17 genera (9 g-positive and 8 g-negative) and 33 differernt species. Overall, 445 (93%) were putatively identified to the genus level by MALDI-TOF MS and 355 (74%) were identified to the species level, but no reliable identification was obtained for 16 (3.3%), and 20 (4.2%) discordant results were identified. Future studies may help to overcome the limitations of the MALDI database and additional sample preparation steps might help to reduce the number of discordances in identification. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional identification methods for common mastitis pathogens, routinely isolated from raw milk. Thus it's adoption will strengthen the capacity, quality, and possibly the scope of diagnostic services to support the dairy industry.

Pneumopericardium causing pericardial tamponade.

Pneumopericardium can be severe enough to cause pericardial tamponade physiology. These patients can be hemodynamically unstable and require pericardial drainage.

Cohort Study of Albumin versus Lactated Ringer's for Postoperative Cardiac Surgery Fluid Resuscitation in the Intensive Care Unit.

PURPOSE: A new postcardiac surgery fluid resuscitation strategy was implemented in our cardiovascular intensive care unit (CVICU) to implement evidence-based practice. We transitioned from a primarily albumin fluid-based strategy to a lactated Ringer's fluid-based strategy. We sought to determine whether a new postoperative fluid resuscitation strategy significantly altered the fluid composition for postcardiac surgery patients and what effect that would have on fluid resuscitation costs. Secondary outcomes included various clinical parameters. METHODS: This was a retrospective, before-and-after cohort study of postcardiac surgery patients in an academic quaternary care intensive care unit (ICU) during two different 3-month time intervals. A total of 192 patients were studied: 108 pre-intervention and 84 post intervention. The intervention consisted of surveying stakeholders regarding potential concerns of reducing albumin use, an educational intervention addressing those concerns, and removing albumin from the routine postcardiac surgery ICU admission order set. RESULTS: In the post intervention time period, albumin use decreased significantly compared to pre-invention (p<0.01), and lactated Ringer's volume increased significantly (p<0.01). However, total volume administered for resuscitation was not significantly different pre- and post intervention (1129 ml vs. 1369 ml, p=0.136). There were a net-cost savings between the pre-intervention and post intervention period (3 mo) of $30,549.20, with the albumin reduction accounting for most of those savings. Secondary outcomes were not significantly different between groups. CONCLUSIONS: An albumin fluid reduction strategy was successful in reducing the amount of albumin fluid used for postcardiac surgery patients and resulted in substantial cost savings.

iTRAQ-Based Global Proteomic Analysis of Salmonella enterica Serovar Typhimurium in Response to Desiccation, Low Water Activity, and Thermal Treatment.

In this study, the changes in the global proteome of Salmonella in response to desiccation and thermal treatment were investigated by using an iTRAQ multiplex technique. A Salmonella enterica serovar Typhimurium strain was dried, equilibrated at high (1.0) and low (0.11) water activity (aw), and thermally treated at 75°C. The proteomes were characterized after every treatment. The proteomes of the different treatments differed in the expression of 175 proteins. On the basis of their proteomic expression profiles, the samples were clustered into two major groups, namely, "dry" samples and "moist" samples. The groups had different levels of proteins involved in DNA synthesis and transcription and in metabolic reactions, indicating that cells under either of the aw conditions need to strictly control energy metabolism, the rate of replication, and protein synthesis. The proteins with higher expression levels in moist samples were flagellar proteins (FlgEFGH), membrane proteins, and export systems (SecF, SecD, the Bam complex), as well as stress response proteins, suggesting that rehydration can trigger stress responses in moist cells. Dry samples had higher levels of ribosomal proteins, indicating that ribosomal proteins might be important for additional regulation of the cellular response, even when the synthesis of proteins is slowed down. At both aws, no differences in protein expression were observed between the thermally treated samples and the nonheated cells. In conclusion, our study indicates that the preadaptation to a dry condition was linked to increased thermal tolerance, while reversion from a dry state to a moist state induced a significant change in protein expression, possibly linked to the observed loss of thermal tolerance.IMPORTANCESalmonella enterica is able to survive in dry environments for very long periods. While it is well known that the initial exposure to desiccation is fundamental to trigger thermal tolerance in this organism, the specific physiological and molecular processes involved in this cross-protection phenomenon have not been fully characterized. Several studies have focused on the low-aw transcriptome of this pathogen when inoculated in different food matrices or on abiotic surfaces, but proteomic analyses have not been reported in the literature. Our study investigated the changes in proteomic expression in Salmonella enterica serovar Typhimurium during desiccation, exposure to low aw, and thermal treatment. A better knowledge of the systems involved in the response to desiccation and thermal tolerance, as well as a better understanding of their interplay, is fundamental to identify the most effective combination of interventions to prevent Salmonella's contamination of foods.

General response of Salmonella enterica serovar Typhimurium to desiccation: A new role for the virulence factors sopD and sseD in survival.

Salmonella can survive for long periods under extreme desiccation conditions. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular regulation of this adaptation mechanism. To determine the genetic components involved in Salmonella's cellular response to desiccation, we performed a global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low water activity (aw 0.11) and cells equilibrated to high water activity (aw 1.0). The analysis revealed that 719 genes were differentially regulated between the two conditions, of which 290 genes were up-regulated at aw 0.11. Most of these genes were involved in metabolic pathways, transporter regulation, DNA replication/repair, transcription and translation, and, more importantly, virulence genes. Among these, we decided to focus on the role of sopD and sseD. Deletion mutants were created and their ability to survive desiccation and exposure to aw 0.11 was compared to the wild-type strain and to an E. coli O157:H7 strain. The sopD and sseD mutants exhibited significant cell viability reductions of 2.5 and 1.3 Log (CFU/g), respectively, compared to the wild-type after desiccation for 4 days on glass beads. Additional viability differences of the mutants were observed after exposure to aw 0.11 for 7 days. E. coli O157:H7 lost viability similarly to the mutants. Scanning electron microscopy showed that both mutants displayed a different morphology compared to the wild-type and differences in production of the extracellular matrix under the same conditions. These findings suggested that sopD and sseD are required for Salmonella's survival during desiccation.

Factors influencing the Salmonella internalization into seedpods and whole plants of Arachis hypogaea (L.).

Here we investigated whether Salmonella enterica serovar Typhimurium ATCC 14028 was capable of internalizing in peanut seedpods and plants when exposed to inoculated soil and the edaphic factors that influenced uptake. Intact dry Virginia (DV) and fresh green Virginia (GV) seedpods were exposed to soil containing 6.5 Log (CFU/g) Salmonella under different soil moisture conditions. Internalization of S. Typhimurium into peanut plants germinated in inoculated soil was also examined with and without Bradyrhizobium (Arachis) sp.NC92. Salmonella counts recovered from GV seedpods were on average of 2.0 Log (CFU/pod) less than those recovered from DV seedpods. The internalization in DV pods was only observed at soil water content of 15% or greater in a loamy sand soil. S. Typhimurium was detected inside peanut plant tissues during most testing times. Cells were recovered from stem samples (3.5 Log CFU/g) at greater levels than it was observed for root (2.6 Log CFU/g) and leaf (1.7 Log CFU/g) samples. Overall, recovery of Salmonella from stem, root, and leaf samples were lower when B. NC92 was inoculated on seeds before sowing, but this trend was not significant. Our observations suggest possible routes of contamination of Salmonella into peanut products from soil.

Complete Genome Sequence of NC983, a Live Attenuated Strain of Salmonella enterica Serovar Typhimurium.

Foodborne infections caused by Salmonella enterica serovars are a significant problem worldwide. Presented here is the genome sequence of the nontyphoidal S. enterica serovar Typhimurium mutant strain NC983, a potential vaccine candidate.

Enteric pathogen-plant interactions: molecular connections leading to colonization and growth and implications for food safety.

Leafy green vegetables have been identified as a source of foodborne illnesses worldwide over the past decade. Human enteric pathogens, such as Escherichia coli O157:H7 and Salmonella, have been implicated in numerous food poisoning outbreaks associated with the consumption of fresh produce. An understanding of the mechanisms responsible for the establishment of pathogenic bacteria in or on vegetable plants is critical for understanding and ameliorating this problem as well as ensuring the safety of our food supply. While previous studies have described the growth and survival of enteric pathogens in the environment and also the risk factors associated with the contamination of vegetables, the molecular events involved in the colonization of fresh produce by enteric pathogens are just beginning to be elucidated. This review summarizes recent findings on the interactions of several bacterial pathogens with leafy green vegetables. Changes in gene expression linked to the bacterial attachment and colonization of plant structures are discussed in light of their relevance to plant-microbe interactions. We propose a mechanism for the establishment and association of enteric pathogens with plants and discuss potential strategies to address the problem of foodborne illness linked to the consumption of leafy green vegetables.

Impact of management practices and distillers' grains feeding on the prevalence of Escherichia coli O157 in feedlot cattle in Minnesota.

Escherichia coli O157 is a foodborne pathogen that can be transmitted by contaminated ground beef and is shed naturally in cattle feces. Recent reports indicated that feeding distillers' grains (DG) to cattle increased fecal shedding and prevalence of E. coli O157. In Minnesota, feeding DG with solubles (DGS) to livestock became widespread within the last 10 years, but there is no report about the prevalence of E. coli O157 in beef cattle in this state. This study was undertaken to survey the fecal prevalence of E. coli O157 in cattle fed diets containing DG and its association with environmental conditions and management practices. Fecal samples were collected from three feedlots during a 1-year period. All animals in those feedlots were fed different DGS levels. E. coli O157 presence was determined using a combination of enrichment, immunomagnetic separation, plating onto sorbitol MacConkey agar, and confirmation of isolates by immunoassay and multiplex virulence genes polymerase chain reaction analysis. Overall, E. coli O157 was confirmed in 9.7% of samples. Prevalence during summer was 30% and declined to less than 10% the rest of the year. In animals grouped by dietary DGS concentration, no significant difference in prevalence (12.0 and 5.5%) was detected between the low and the high average groups (less and more than 20%). Previous feeding of DGS before arriving to the feedlot also had no influence on fecal prevalence. The presence of several interacting variables, uncontrolled in a real-life feedlot environment, was the likely reason for our observation and suggested that at the levels studied, DGS had no effect on the STEC O157 prevalence in cattle populations.

Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane.

Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.

Incidence of naturally internalized bacteria in lettuce leaves.

Lettuce is the fresh leafy vegetable most frequently involved in foodborne disease outbreaks. Human bacterial pathogens may be experimentally internalized into lettuce plants, but the occurrence of natural microflora inside lettuce leaves has not been elucidated. To characterize the endophytic microorganism residing in commercial lettuce leaves, two separate studies were conducted. First, a total of 30 and 25 heads of romaine and red leaf lettuce, respectively, served as the source of individual leaves which were surface sterilized, stomached, enriched in BHI broth for 24h and plated onto BHI agar for non-selective isolation of internalized microorganism. In a separate survey, 80 heads of each of the two types of lettuce were similarly processed, except that GN broth and MacConkey agar (MCA) were used for isolation of Gram negative bacteria. Thirty-eight out of 100 leaves were positive for internalized microorganisms, and Bacillus, Pseudomonas and Pantoea were the genera most frequently found in both types of lettuce. Members of the genus Erwinia were isolated from romaine lettuce only. In the second study, 21 and 60% of romaine and red leaf lettuce heads, respectively, had internalized bacteria capable of growing on MCA. Among the Gram negative strains, Pseudomonas and Pantoea genera were most frequently isolated. Enterobacter isolates were obtained from three red leaf samples. In summary, spore-forming bacteria and traditional epiphytic bacterial genera were frequently detected in surface-sterilized commercial lettuce leaves. Despite the common occurrence of internalized bacteria, only Enterobacter was related to Escherichia coli O157:H7 and Salmonella.

Transcriptional responses of Escherichia coli K-12 and O157:H7 associated with lettuce leaves.

An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.

Optimized turmeric extract reduces ß-Amyloid and phosphorylated Tau protein burden in Alzheimer's transgenic mice.

In a previous in vitro study, the standardized turmeric extract, HSS-888, showed strong inhibition of Aß aggregation and secretion in vitro, indicating that HSS-888 might be therapeutically important. Therefore, in the present study, HSS-888 was evaluated in vivo using transgenic 'Alzheimer' mice (Tg2576) over-expressing Aß protein. Following a six-month prevention period where mice received extract HSS-888 (5mg/mouse/day), tetrahydrocurcumin (THC) or a control through ingestion of customized animal feed pellets (0.1% w/w treatment), HSS-888 significantly reduced brain levels of soluble (∼40%) and insoluble (∼20%) Aß as well as phosphorylated Tau protein (∼80%). In addition, primary cultures of microglia from these mice showed increased expression of the cytokines IL-4 and IL-2. In contrast, THC treatment only weakly reduced phosphorylated Tau protein and failed to significantly alter plaque burden and cytokine expression. The findings reveal that the optimized turmeric extract HSS-888 represents an important step in botanical based therapies for Alzheimer's disease by inhibiting or improving plaque burden, Tau phosphorylation, and microglial inflammation leading to neuronal toxicity.