Using Farm Practice Variables as Predictors of Listeria spp. Prevalence in Pastured Poultry Farms

Predictive models offer food scientists, farmers, and processors tools to help identify variables that lead to an increase in the food safety risk of a product. Foodborne pathogens, such as Listeria spp., pose a major problem for the pastured poultry industry. Currently, there is a lack of understanding of what farm practices lead to higher prevalence of Listeria spp. This study constructed random forest (RF) models to predict the prevalence of Listeria spp. in pastured poultry farming environments and the final broiler product based on major farm practices and variables. Feces, soil, and whole carcass rinse samples were collected from 11 farms in the southeastern United States and evaluated for Listeria spp. presence. The preharvest sample RF model identified the time of year and age of the broiler flock at time of sampling as factors of increased probability of Listeria spp. presence in feces and soil samples. The final product RF model identified brood feed and the presence of chlorine in processing rinse water as the two most important variables associated with an increased likelihood of Listeria spp. presence. Both the preharvest RF model and final sample RF model performed well on a held-out test set, with area under the receiver operating characteristic curve values of 0.876 and 0.887, respectively. The presented models showed the usefulness of RF models in a food safety context. Both RF models will help pastured poultry farmers and processors guide control strategies to manage Listeria contamination in pastured poultry farms and products

The effect of lauric arginate on the thermal inactivation of starved Listeria monocytogenes in sous-vide cooked ground beef

The aim of this study was to examine the efficacy of lauric arginate (LAE, 1000 ppm – 3000 ppm) as an assisting tool to reduce starved Listeria monocytogenes population in ground beef following sous-vide processing at different temperatures (55–62.5 °C). Ground beef mixed with LAE was vacuum sealed and a laboratory water bath was used for sous-vide cooking. Loglinear and Weibull models were fit to the survival microbial population and the D and Z-values were determined at 55–62.5 °C. Calculated D-values ranged from 33.62 to 3.22 min at temperature 55–62.5 °C. LAE at higher concentration is an effective antimicrobial to increase the inactivation of the pathogen in sous-vide cooking. With the addition of LAE, D-values at 55 and 62.5 °C determined by the Loglinear model decreased from 31.86 to 2.28 min (LAE 1000 ppm) and 16.71 to 0.56 min (LAE 3000 ppm), respectively; whereas the D-values at 55 to 62.5 °C determined by the Weibull model were 44.26 and 2.09 min (LAE 1000 ppm) and 22.71 and 1.60 min (LAE 3000 ppm), respectively. This study shows that sous-vide processing of ground beef supplemented with higher concentration of LAE effectively inactivates L. monocytogenes and thus, helps increase the microbiological safety and product quality

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

Choosing and Using a Plant DNA Barcode

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance

A Survey of New Temperature-Sensitive, Embryonic-Lethal Mutations in C. elegans: 24 Alleles of Thirteen Genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci

The management of diabetic ketoacidosis in children

The object of this review is to provide the definitions, frequency, risk factors, pathophysiology, diagnostic considerations, and management recommendations for diabetic ketoacidosis (DKA) in children and adolescents, and to convey current knowledge of the causes of permanent disability or mortality from complications of DKA or its management, particularly the most common complication, cerebral edema (CE). DKA frequency at the time of diagnosis of pediatric diabetes is 10%–70%, varying with the availability of healthcare and the incidence of type 1 diabetes (T1D) in the community. Recurrent DKA rates are also dependent on medical services and socioeconomic circumstances. Management should be in centers with experience and where vital signs, neurologic status, and biochemistry can be monitored with sufficient frequency to prevent complications or, in the case of CE, to intervene rapidly with mannitol or hypertonic saline infusion. Fluid infusion should precede insulin administration (0.1 U/kg/h) by 1–2 hours; an initial bolus of 10–20 mL/kg 0.9% saline is followed by 0.45% saline calculated to supply maintenance and replace 5%–10% dehydration. Potassium (K) must be replaced early and sufficiently. Bicarbonate administration is contraindicated. The prevention of DKA at onset of diabetes requires an informed community and high index of suspicion; prevention of recurrent DKA, which is almost always due to insulin omission, necessitates a committed team effort

Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex

Mapping foodborne pathogen contamination throughout the conventional and alternative poultry supply chains

ABSTRACT: Recently, there has been a consumer push for natural and organic food products. This has caused alternative poultry production, such as organic, pasture, and free-range systems, to grow in popularity. Due to the stricter rearing practices of alternative poultry production systems, different types of levels of microbiological risks might be present for these systems when compared to conventional production systems. Both conventional and alternative production systems have complex supply chains that present many different opportunities for flocks of birds or poultry meat to be contaminated with foodborne pathogens. As such, it is important to understand the risks involved during each step of production. The purpose of this review is to detail the potential routes of foodborne pathogen transmission throughout the conventional and alternative supply chains, with a special emphasis on the differences in risk between the two management systems, and to identify gaps in knowledge that could assist, if addressed, in poultry risk-based decision making

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light-dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light–dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day