PCR-ELISAs for the detection of Campylobacter jejuni and Campylobacter coli in poultry samples

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on Nucleo-Link wells and hybridized to PCR products modified with a 5' biotin moiety. The limit of detection of the PCR-ELISA was 100-300 fg (40-120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.     

Tyrosine availability prevents tyramine-induced tachyphylaxis in the isolated rat heart

Isolated rat hearts perfused with Chenoweth-Koelle solution exhibit tachyphylaxis to tyramine. This tachyphylaxis was prevented if tyrosine was included in the perfusion solution. Other amino acids, including glycine, serine, phenylalanine, valine, tryptophan, glutamate, aspartate, arginine, lysine and -tyrosine, failed to prevent the tyramine-induced tachyphylaxis. Hearts from reserpinized animals showed increased chronotropy after tyramine only when tyrosine was present in the medium. This response could be blocked by , m-hydroxybenzyl-hydrazine and d,l-propanolol, indicating that it was mediated by the synthesis and release of catecholamines. These experiments suggest that sympathetic nerves in the isolated rat heart exhibit a requirement for tyrosine when catecholamine release is enhanced.     

Problems with the use of terracotta clay saucers as phosphorus-diffusing substrata to assess nutrient limitation of epilithic algae

1. We examined the diffusion properties of terracotta clay saucers, of types often used as components in phosphorus-diffusing substrata for investigating nutrient limitation of epilithic algae.
2. Laboratory experiments showed that phosphorus diffusion was low and inconsistent (0.06–2.6 mg P day⁻¹) through clay saucers filled with agar containing orthophosphate. Similarly, in situ release of phosphorus from two types of terracotta clay saucers (Australian and Italian) was variable (2–8 mg P day⁻¹; 5–25%) under three flow regimes (0, 0.1 and 0.3 m s⁻¹) over 30 days, with most phosphorus being released during the first day. Clogging of pores by agar appears to prevent the diffusion of phosphorus through the terracotta clay saucers. However, the two types of terracotta clay saucer also irreversibly sorbed large quantities of phosphorus (40–140 mg P).
3. Individual saucers can have markedly different physical and chemical properties both within and among terracotta types. Terracotta saucers also contain large quantities of iron, calcium and aluminium that are known binding agents for phosphorus. Variability in saucer composition and diffusion properties makes treatments difficult to replicate and weakens comparisons between studies that have used terracotta with different clay composition and manufacture.
4. We recommend that phosphorus-diffusing substrata should not be constructed from terracotta clay components.     

The presence of arsenobetaine in marine animals

Methanol soluble arsenic compounds were isolated from the muscle tissues of a crayfish, Jasus novaehollandiae, a prawn, Penaeus latisulcatus, a scallop, Pecten alba, a squid, Sepioteuthis australis and a fish, Sillaginodes punctatus. Arsenobetaine was found to be the major arsenic compound present in all muscle tissues. The prawn and scallop tissues also contained another organo arsenic compound that could be partially degraded to characterized methylated arsenic species.     

Mutations to azaguanine resistance induced in cultured diploid human fibroblasts by the carcinogen, N-acetoxy-2-Acetylaminofluorene

The ability of the carcinogen, N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), to induce mutations to azaguanine resistance in diploid human cells was quantitatively investigated and shown to be dose-dependent. The 8-azaguanine (AG) resistance was shown to be heritable in the absence of mutagen or selective agent and the cells of the mutant clones were shown to retain normal sensitivity to N-AcO-AAF.     

Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

Benefiting others and self: Production of vitamins in plants

Vitamins maintain growth and development in humans, animals, and plants. Because plants serve as essential producers of vitamins, increasing the vitamin contents in plants has become a goal of crop breeding worldwide. Here, we begin with a summary of the functions of vitamins. We then review the achievements to date in elucidating the molecular mechanisms underlying how vitamins are synthesized, transported, and regulated in plants. We also stress the exploration of variation in vitamins by the use of forward genetic approaches, such as quantitative trait locus mapping and genome-wide association studies. Overall, we conclude that exploring the diversity of vitamins could provide new insights into plant metabolism and crop breeding.     

Chemotherapy regimens induce inhibitory immune checkpoint protein expression on stem-like and senescent-like oesophageal adenocarcinoma cells

Use of immune checkpoint inhibitors (ICIs) with chemotherapy to enhance responses in oesophageal adenocarcinoma (OAC) is an attractive approach. We identified subpopulations of OAC cells expressing inhibitory immune checkpoint (IC) ligands (PD-L1, PD-L2 and CD160) and receptors (PD-1, TIGIT, TIM-3, LAG-3 and A2aR) in vitro and in ex vivo biopsies. Combination chemotherapy regimens FLOT and CROSS promote a more immune-resistant phenotype through upregulation of IC ligands and receptors on OAC cells in vitro. Importantly, this study investigated if OAC cells, enriched for ICs exhibited a more stem-like and senescent-like phentoype. FLOT preferentially upregulates PD-L1 on a stem-like OAC cell phenotype, defined by ALDH activity. Expression of senescence-associated β-galactosidase is induced in a subpopulation of OAC cells following FLOT and CROSS chemotherapy treatment, along with enhanced expression of TIM-3 and A2aR ICs. Blockade of PD-1 signalling in OAC cells induced apoptosis and enhanced FLOT and CROSS chemotherapy toxicity in vitro. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance. Combination ICIs may be required to enhance the efficacy of chemotherapy and immunotherapy in OAC patients.     

Redox status in workers occupationally exposed to long-term low levels of ionizing radiation: A pilot study

Objectives: Reactive oxygen species (ROS), including superoxide (O₂^??), play an important role in the biological effects of ionizing radiation. The human body has developed different antioxidant systems to defend against excessive levels of ROS. The aim of the present study is to investigate the redox status changes in the blood of radiologic technologists and compare these changes to control individuals.

Methods: We enrolled 60 medical workers: 20 occupationally exposed to ionizing radiation (all radiologic technologists), divided in three subgroups: conventional radiography (CR), computerized tomography (CT), and interventional radiography (IR) and 40 age- and gender-matched unexposed controls. Levels of O₂^?? and malondialdehyde (MDA) in blood were measured as an index of redox status, as were the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase. Redox status was also assessed by measuring levels of reduced and oxidized glutathione (GSH, GSSG, respectively).

Results: Levels of O₂^?? and MDA, and SOD activity in the blood of IR and CT-exposed subjects were significantly higher than both the CR-exposed subjects and control individuals. However, there were no statistically significant differences in the levels of catalase, GSH and ratio of GSH/GSSG between exposed workers and control individuals.

Discussion: This study suggests that healthcare workers in CT and IR occupationally exposed to radiation have an elevated circulating redox status as compared to unexposed individuals.