Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Molecular effects of a variable environment on Sydney rock oysters, Saccostrea glomerata: thermal and low salinity stress, and their synergistic effect

Bivalves are frequently exposed to salinity and temperature fluctuations in the estuary. This study explored the molecular effect of these fluctuations by exposing Sydney rock oysters, (Saccostrea glomerata), native to Australia, to either low salinity, elevated temperature or a combined salinity and temperature stress. Following the exposures, RNA-Seq was carried out on the collected oyster tissues. Differential transcript analysis resulted in a total of 1473, 1232 and 2571 transcripts, which were differentially expressed in S. glomerata exposed to low salinity (10 ppt), elevated temperature (30 °C) or the combined stressor (15 ppt and 30 °C), respectively, when compared to control oysters. All stress treatments had some effect on molecular processes such as innate immune response or respiration, with overall the strongest effects seen in S. glomerata exposed to the combined stressor. Additionally, most transporters putatively involved in osmoregulation were found to be suppressed in response to the combined stressor and the low salinity exposure. This study provides insight into the oyster's responses to both, single and dual stressors commonly found in an estuarine environment

Molecular analysis of the Sydney rock oyster (Saccostrea glomerata) CO2 stress response

Background Human activities have led to a substantial increase in carbon dioxide (CO2) emission, with further increases predicted. A RNA-Seq study on adult Saccostrea glomerata was carried out to examine the molecular response of this bivalve species to elevated pCO2. Results A total of 1626 S. glomerata transcripts were found to be differentially expressed in oysters exposed to elevated pCO2 when compared to control oysters. These transcripts cover a range of functions, from immunity (e.g. pattern recognition receptors, antimicrobial peptides), to respiration (e.g. antioxidants, mitochondrial respiratory chain proteins) and biomineralisation (e.g. carbonic anhydrase). Overall, elevated levels of CO2 appear to have resulted in a priming of the immune system and in producing countermeasures to potential oxidative stress. CO2 exposure also seems to have resulted in an increase in the expression of proteins involved in protein synthesis, whereas transcripts putatively coding for proteins with a role in cilia and flagella function were down-regulated in response to the stressor. In addition, while some of the transcripts related to biomineralisation were up-regulated (e.g. carbonic anhydrase 2, alkaline phosphatase), a small group was down-regulated (e.g. perlucin). Conclusions This study highlighted the complex molecular response of the bivalve S. glomerata to expected near-future ocean acidification levels. While there are indications that the oyster attempted to adapt to the stressor, gauged by immune system priming and the increase in protein synthesis, some processes such cilia function appear to have been negatively affected by the elevated levels of CO2

Combined exposure to pyrene and fluoranthene and their molecular effects on the Sydney rock oyster, Saccostrea glomerata

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously detected in the water column, associated with particulate matter or in the tissue of marine organisms such as molluscs. PAH exposure and their resultant bioaccumulation in molluscs can cause a range of serious physiological effects in the affected animals. To examine the molecular response of these xenobiotics in bivalves, Sydney rock oysters (Saccostrea glomerata) were exposed to pyrene and fluoranthene for seven days. Chemical analysis of the soft-tissue of PAH stressed S. glomerata confirmed that pyrene and fluoranthene could be bioaccumulated by these oysters. RNA-Seq analysis of PAH-exposed S. glomerata showed a total of 765 transcripts differentially expressed between control and PAH-stressed oysters. Closer examination of the transcripts revealed a range genes encoding enzymes involved in PAH detoxification (e.g. cytochrome P450), innate immune responses (e.g. pathogen recognition, phagocytosis) and protein synthesis. Overall, pyrene and fluoranthene exposure appears to have resulted in a suppression of pathogen recognition and some protein synthesis processes, whereas transcripts of genes encoding proteins involved in clearance of cell debris and some transcripts of genes involved in PAH detoxification were induced in response to the stressors. Pyrene and fluoranthene exposure thus invoked a complex molecular response in S. glomerata, with results suggesting that oysters focus on removing the stressors from their system and dealing with the downstream effects of PAH exposure, potentially at the exclusion of other, less immediate concerns (e.g. protection from infection)

Transcriptome analysis of the Sydney rock oyster, Saccostrea glomerata: insights into molluscan immunity

Background: Oysters have important ecological functions in their natural environment, acting as global carbon sinks and improving water quality by removing excess nutrients from the water column. During their life-time oysters are exposed to a variety of pathogens that can cause severe mortality in a range of oyster species. Environmental stressors encountered in their habitat can increase the susceptibility of oysters to these pathogens and in general have been shown to impact on oyster immunity, making immune parameters expressed in these marine animals an important research topic. Results: Paired-end Illumina high throughput sequencing of six S. glomerata tissues exposed to different environmental stressors resulted in a total of 484,121,702 paired-end reads. When reads and assembled transcripts were compared to the C. gigas genome, an overall low level of similarity at the nucleotide level, but a relatively high similarity at the protein level was observed. Examination of the tissue expression pattern showed that some transcripts coding for cathepsins, heat shock proteins and antioxidant proteins were exclusively expressed in the haemolymph of S. glomerata, suggesting a role in innate immunity. Furthermore, analysis of the S. glomerata ORFs showed a wide range of genes potentially involved in innate immunity, from pattern recognition receptors, components of the Toll-like signalling and apoptosis pathways to a complex antioxidant defence mechanism. Conclusions: This is the first large scale RNA-Seq study carried out in S. glomerata, showing the complex network of innate immune components that exist in this species. The results confirmed that many of the innate immune system components observed in mammals are also conserved in oysters; however, some, such as the TLR adaptors MAL, TRIF and TRAM are either missing or have been modified significantly. The components identified in this study could help explain the oysters' natural resilience against pathogenic microorganisms encountered in their natural environment

Schematic of the predicted innate immune processes of <i>S</i>. <i>glomerata</i>.

<p><i>S</i>. <i>glomerata</i> innate immune transcripts observed in this study are predicted to be involved in a range of different innate immune processes, (components regulating the different pathways are not visualised in this figure). Abbreviations are as follows: PPRs (pattern recognition receptors), TLR (Toll-like receptor), PAMPs (pathogen-associated molecular patterns), DAMPs (damage-associated molecular patterns), TRAF (tumor necrosis factor receptor-associated factor), TAK1 (transforming-growth-factor-β-activated kinase 1), ROS (reactive oxygen species), ECSIT (evolutionary conserved signalling intermediate in toll pathways), RIG-1/MDA5 (interferon-induced helicase c domain-containing protein 1), MAVS (mitochondrial antiviral-signalling protein), AIF (apoptosis-inducing factor 1), AP-1 (activator protein 1), NF-κB (nuclear factor kappa B), IRF (interferon regulatory factor), HIF (hypoxia-inducible factor), CBP (CREB-binding protein), RNI (reactive nitrogen intermediates), AMP (antimicrobial peptide). ↑ arrows used in the figure (excluding the ones used in the nucleus) depict signalling pathways.</p

Transcript patterns across six different <i>S</i>. <i>glomerata</i> tissues.

<p><b>A)</b> non-normalised and non-strand specific tissue reads were mapped to the combined reference transcriptome and <b>b)</b> strand-specific and normalised tissue reads were mapped to the strand-specific reference transcriptome, using CLC Workbench version 7.5. Graph shows the number of transcripts that are common across individual tissue patterns, with 1 standing for “transcript present in tissue” and 0 for “transcript not present in tissue”. The order in which the tissues are listed is as follows: haemolymph (closest to the x-axis label), gill, mantle, muscle, gonad and digestive. The respective top graphs show the full-scale graph, with the respective lower graphs emphasising the fine scale features of the same graph by adding a graph break.</p

Potential <i>S</i>. <i>glomerata</i> immune and immune related ORFs determined by GO and/or InterProScan annotation.

<p>Potential <i>S</i>. <i>glomerata</i> immune and immune related ORFs determined by GO and/or InterProScan annotation.</p

Summary of <i>S</i>. <i>glomerata</i> assembly, ORF prediction and CEGMA analysis.

<p>Summary of <i>S</i>. <i>glomerata</i> assembly, ORF prediction and CEGMA analysis.</p