QHFSS DNA laboratory - ISO/IEC 17025 conformance and accreditation.

This paper reviews evidence placed before a Commission of Inquiry (CoI) established by the State of Queensland, Australia, to consider the quality and reliability of DNA evidence. It also assesses whether the criticism levied in that report, of ISO/IEC 17025 being insufficient to assure the quality and reliability of DNA evidence, is warranted. The main conclusion drawn is that properly applied and embraced as a means of continuous improvement, conformance with ISO/IEC 17025:2017 alone is sufficient to assure the quality and reliability of the scientific outputs from a forensic science laboratory. Furthermore, it is clear from the observations and findings of the CoI and those recorded in this paper that the forensic science laboratory in question did not conform to ISO/IEC 17025:2017. Had it done so then the risk of the quality failures that led to the CoI would at least have been reduced and perhaps even avoided.

IRMS based evidence passes the test.

At the two latest FIRMS (The Forensic Isotope Ratio Mass Spectrometry Network Ltd) conferences, presentations were made describing the status of IRMS based evidence in criminal proceedings [S. Doyle, T. Chau and J. Howa, IRMS based evidence passes the 'test' (aided by FIRMS) - an important milestone, in: 8th FIRMS Conference, 2022; J. Ehleringer, J. Howa and T. Chau, Stable isotope evidence in the U.S. courts, in: 7th FIRMS Conference, 2019]. One of those cases was of particular significance in that it established the admissibility of IRMS (isotope ratio mass spectrometry) based expert testimony having satisfied the US Federal Rule of Evidence (FRE)702 'Testimony by Expert Witnesses' in the State of Colorado. While specific to the State of Colorado and the particulars of the case, the admissibility of IRMS based evidence in other cases might be supported by such a ruling. This paper provides the detail of the process by which IRMS based evidence was found to be admissible and in so doing also provides practical guidance on how to satisfy FRE 702 and meet a Daubert challenge. It also highlights the role of approval by the FIRMS Network of both practitioners and their methods in support of admissibility [The FIRMS Network Ltd., FIRMS Approved Practitioners, [Online]. Available: https://www.forensic-isotopes.org/fafp.html [Accessed 11 May 2023].].

The use of cathepsin L1 (FhCL1) serological ELISA in sentinel screening for liver fluke on sheep farms.

Fasciola hepatica is a parasitic helminth (worm) that poses a significant economic threat to the ruminant livestock industry worldwide. The disease, fasciolosis, can result in a range of clinical signs including anaemia, weight loss and death, with the most severe symptoms attributed to early acute infection when the parasite is migrating through the liver. Early diagnosis and intervention are essential for the control and management of the disease to prevent productivity losses. The traditional gold standard method of diagnosis uses faecal egg counts (FEC) that is limited to detecting patent infections from 10 to 12 weeks post infection (WPI). In contrast, serological assays can detect pre-patent infections as we have shown that enzyme-linked immunosorbent assays (ELISA) using the F. hepatica cysteine peptidase cathepsin L1 (FhCL1) can detect liver fluke infections from 3 to 4 WPI. Here, we used FEC and ELISA to monitor liver fluke infections in sentinel lambs from three commercial farms in Ireland from September 2021 to March 2022. All three farms showed a significant increase in FhCL1 antibody levels and FEC over this time, with a substantial rise in positive infection detection between late November and January. However, ELISA screening detected infection at least two months prior to FEC (September). This suggests that the regular screening of sentinel lambs for F. hepatica seroconversion in a "test and treat" approach could mitigate the negative damaging impact of early fasciolosis on flock health, welfare and productivity and inform management strategies. In addition, we show that whole blood samples taken on Whatman® protein saver cards could replace conventional serum blood tubes for blood collection. Cards can be stored at room temperature for long periods of time and samples revisited at any time for re-analysis. The adoption of these cards on farm together with the FhCL1 ELISA would provide a simpler, cost-effective, and eco-friendly method for testing sentinel lambs for liver fluke disease.

Transcriptional profiling of a fungal granuloma reveals a low metabolic activity of Paracoccidioides brasiliensis yeasts and an actively regulated host immune response.

Granulomas are important immunological structures in the host defense against the fungus Paracoccidioides brasiliensis, the main etiologic agent of Paracoccidioidomycosis (PCM), a granulomatous systemic mycosis endemic in Latin America. We have performed transcriptional and proteomic studies of yeasts present in the pulmonary granulomas of PCM aiming to identify relevant genes and proteins that act under stressing conditions. C57BL/6 mice were infected with 1x106 yeasts and after 8- and 12-weeks of infection, granulomatous lesions were obtained for extraction of fungal and murine RNAs and fungal proteins. Dual transcriptional profiling was done comparing lung cells and P. brasiliensis yeasts from granulomas with uninfected lung cells and the original yeast suspension used in the infection, respectively. Mouse transcripts indicated a lung malfunction, with low expression of genes related to muscle contraction and organization. In addition, an increased expression of transcripts related to the activity of neutrophils, eosinophils, macrophages, lymphocytes as well as an elevated expression of IL-1ß, TNF-α, IFN-γ, IL-17 transcripts were observed. The increased expression of transcripts for CTLA-4, PD-1 and arginase-1, provided evidence of immune regulatory mechanisms within the granulomatous lesions. Also, our results indicate iron as a key element for the granuloma to function, where a high number of transcripts related to fungal siderophores for iron uptake was observed, a mechanism of fungal virulence not previously described in granulomas. Furthermore, transcriptomics and proteomics analyzes indicated a low fungal activity within the granuloma, as demonstrated by the decreased expression of genes and proteins related to energy metabolism and cell cycle.

Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion depletion.

Overcoming antimicrobial resistance represents a formidable challenge and investigating bacterial growth inhibition by fungal metabolites may yield new strategies. Although the fungal non-ribosomal peptide gliotoxin (GT) is known to exhibit antibacterial activity, the mechanism(s) of action are unknown, although reduced gliotoxin (dithiol gliotoxin; DTG) is a zinc chelator. Furthermore, it has been demonstrated that GT synergises with vancomycin to inhibit growth of Staphylococcus aureus. Here we demonstrate, without precedent, that GT-mediated growth inhibition of both Gram positive and negative bacterial species is reversed by Zn2+ or Cu2+ addition. Both GT, and the known zinc chelator TPEN, mediate growth inhibition of Enterococcus faecalis which is reversed by zinc addition. Moreover, zinc also reverses the synergistic growth inhibition of E. faecalis observed in the presence of both GT and vancomycin (4 µg/ml). As well as zinc chelation, DTG also appears to chelate Cu2+, but not Mn2+ using a 4-(2-pyridylazo)resorcinol assay system and Zn2+ as a positive control. DTG also specifically reacts in Fe3+-containing Siderotec™ assays, most likely by Fe3+ chelation from test reagents. GSH or DTT show no activity in these assays. Confirmatory high resolution mass spectrometry, in negative ion mode, confirmed, for the first time, the presence of both Cu[DTG] and Fe[DTG]2 chelates. Label free quantitative proteomic analysis further revealed major intracellular proteomic remodelling within E. faecalis in response to GT exposure for 30-180 min. Globally, 4.2-7.2% of detectable proteins exhibited evidence of either unique presence/increased abundance or unique absence/decreased abundance (n = 994-1160 total proteins detected), which is the first demonstration that GT affects the bacterial proteome in general, and E. faecalis, specifically. Unique detection of components of the AdcABC and AdcA-II zinc uptake systems was observed, along with apparent ribosomal reprofiling to zinc-free paralogs in the presence of GT. Overall, we hypothesise that GT-mediated bacterial growth inhibition appears to involve intracellular zinc depletion or reduced bioavailability, and based on in vitro chelate formation, may also involve dysregulation of Cu2+ homeostasis.

Proteomic dissection of the role of GliZ in gliotoxin biosynthesis in Aspergillus fumigatus.

Gliotoxin (GT) biosynthesis in fungi is encoded by the gli biosynthetic gene cluster. While GT addition autoinduces biosynthesis, Zn2+ has been shown to attenuate cluster activity, and it was speculated that identification of Zn2Cys6 binuclear transcription factor GliZ binding partners might provide insight into this observation. Using the Tet-ON induction system, doxycycline (DOX) presence induced GliZ fusion protein expression in, and recovery of GT biosynthesis by, A. fumigatus ΔgliZ::HA-gliZ and ΔgliZ::TAP-gliZ strains, respectively. Quantitative RT-PCR confirmed that DOX induces gli cluster gene expression (n = 5) in both A. fumigatus HA-GliZ and TAP-GliZ strains. GT biosynthesis was evident in Czapek-Dox and in Sabouraud media, however tagged GliZ protein expression was more readily detected in Sabouraud media. Unexpectedly, Zn2+ was essential for GliZ fusion protein expression in vivo, following 3 h DOX induction. Moreover, HA-GliZ abundance was significantly higher in either DOX/GT or DOX/Zn2+, compared to DOX-only. This suggests that while GT induction is still intact, Zn2+ inhibition of HA-GliZ production in vivo is lost. Co-immunoprecipitation revealed that GT oxidoreductase GliT associates with GliZ in the presence of GT, suggesting a potential protective role. Additional putative HA-GliZ interacting proteins included cystathionine gamma lyase, ribosomal protein L15 and serine hydroxymethyltransferase (SHMT). Total mycelial quantitative proteomic data revealed that GliT and GtmA, as well as several other gli cluster proteins, are increased in abundance or uniquely expressed with GT addition. Proteins involved in sulphur metabolism are also differentially expressed with GT or Zn2+ presence. Overall, we disclose that under DOX induction GliZ functionality is unexpectedly evident in zinc-replete media, subject to GT induction and that GliT appears to associate with GliZ, potentially to prevent dithiol gliotoxin (DTG)-mediated GliZ inactivation by zinc ejection.

Aspergillus fumigatus Supernatants Disrupt Bronchial Epithelial Monolayers: Potential Role for Enhanced Invasion in Cystic Fibrosis.

Aspergillus fumigatus is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of A. fumigatus colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of A. fumigatus supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o-) and HBE (16HBE14o-) trans-epithelial electrical resistance (TEER) was measured following exposure to A. fumigatus reference and clinical isolates, a gliotoxin-deficient mutant (ΔgliG) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. A. fumigatus conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while ΔgliG mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by A. fumigatus supernatants but not by ΔgliG supernatants, suggesting that gliotoxin is involved in this process. The fact that ΔgliG conidia were still capable of disrupting epithelial monolayers indicates that direct cell-cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.

Gliotoxin and related metabolites as zinc chelators: implications and exploitation to overcome antimicrobial resistance.

Antimicrobial resistance (AMR) is a major global problem and threat to humanity. The search for new antibiotics is directed towards targeting of novel microbial systems and enzymes, as well as augmenting the activity of pre-existing antimicrobials. Sulphur-containing metabolites (e.g., auranofin and bacterial dithiolopyrrolones [e.g., holomycin]) and Zn2+-chelating ionophores (PBT2) have emerged as important antimicrobial classes. The sulphur-containing, non-ribosomal peptide gliotoxin, biosynthesised by Aspergillus fumigatus and other fungi exhibits potent antimicrobial activity, especially in the dithiol form (dithiol gliotoxin; DTG). Specifically, it has been revealed that deletion of the enzymes gliotoxin oxidoreductase GliT, bis-thiomethyltransferase GtmA or the transporter GliA dramatically sensitise A. fumigatus to gliotoxin presence. Indeed, the double deletion strain A. fumigatus ΔgliTΔgtmA is especially sensitive to gliotoxin-mediated growth inhibition, which can be reversed by Zn2+ presence. Moreover, DTG is a Zn2+ chelator which can eject zinc from enzymes and inhibit activity. Although multiple studies have demonstrated the potent antibacterial effect of gliotoxin, no mechanistic details are available. Interestingly, reduced holomycin can inhibit metallo-ß-lactamases. Since holomycin and gliotoxin can chelate Zn2+, resulting in metalloenzyme inhibition, we propose that this metal-chelating characteristic of these metabolites requires immediate investigation to identify new antibacterial drug targets or to augment the activity of existing antimicrobials. Given that (i) gliotoxin has been shown in vitro to significantly enhance vancomycin activity against Staphylococcus aureus, and (ii) that it has been independently proposed as an ideal probe to dissect the central 'Integrator' role of Zn2+ in bacteria - we contend such studies are immediately undertaken to help address AMR.

A new multiplex SARS-CoV-2 antigen microarray showed correlation of IgG, IgA, and IgM antibodies from patients with COVID-19 disease severity and maintenance of relative IgA and IgM antigen binding over time.

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity different from the same isotype in whole serum, probably due to competition from the other isotypes present. Using purified antibody isotypes from naïve Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with binding to the S region S1 expressed in insect cells (S1 Sf21) significant for IgG, IgA, and IgM. Assessing longitudinal response for constant concentrations of purified antibody isotypes for a patient subset demonstrated that the relative proportion of antigen-specific IgGs decreased over time for severe disease, but the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen-specific serum IgA and IgM playing a role in maintaining longer-term protection, important for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

Target directed identification of natural bioactive compounds from filamentous fungi.

Some of the most powerful natural antimicrobial compounds originate from filamentous fungi. However, due to the diversity of compounds from plant and fungal origin, separation, isolation, and identification of bioactive constituents can be a long and tedious process. This study explores the effectiveness of thin layer chromatography (TLC) in combination with bioautography in the separation and identification of bioactive compounds from several filamentous fungi. Ultra-performance liquid chromatography coupled to photodiode array detector (UPLC-DAD) was employed to quantitatively identify phenolic composition. The total phenolic content of the selected filamentous fungi ranged from 31.85 mg g-1 to 101.77 mg g-1. Additionally, liquid chromatography mass spectrometry (LC/MS) determined the most abundant fatty acids were linoleic, palmitic, oleic, and stearic acid. Submerged cultivation of Grifola frondosa, Monascus purpureus, Lentinula edodes, Trametes versicolor and Pleurotus ostreatus proved to be an effective method to produce natural antimicrobial compounds.

Quantitative Proteomic Analysis Reveals Yeast Cell Wall Products Influence the Serum Proteome Composition of Broiler Chickens.

With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer selected beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW-based Gut Health Products (GHP) or a control basal diet. Development of a novel reagent enabled application of ProteoMiner™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p < 0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, GHP-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with GHP supplementation. Several effects of these GHPs including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase and catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one GHP feed supplement, were observed. It is proposed that specific protein detection may be indicative of GHP efficacy to stimulate broiler immune status, i.e., may be biomarkers of GHP efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that GHP supplementation has on the broiler chicken.

A Single Aspergillus fumigatus Gene Enables Ergothioneine Biosynthesis and Secretion by Saccharomyces cerevisiae.

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC−mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use.

Highdicom: a Python Library for Standardized Encoding of Image Annotations and Machine Learning Model Outputs in Pathology and Radiology.

Machine learning (ML) is revolutionizing image-based diagnostics in pathology and radiology. ML models have shown promising results in research settings, but the lack of interoperability between ML systems and enterprise medical imaging systems has been a major barrier for clinical integration and evaluation. The DICOM® standard specifies information object definitions (IODs) and services for the representation and communication of digital images and related information, including image-derived annotations and analysis results. However, the complexity of the standard represents an obstacle for its adoption in the ML community and creates a need for software libraries and tools that simplify working with datasets in DICOM format. Here we present the highdicom library, which provides a high-level application programming interface (API) for the Python programming language that abstracts low-level details of the standard and enables encoding and decoding of image-derived information in DICOM format in a few lines of Python code. The highdicom library leverages NumPy arrays for efficient data representation and ties into the extensive Python ecosystem for image processing and machine learning. Simultaneously, by simplifying creation and parsing of DICOM-compliant files, highdicom achieves interoperability with the medical imaging systems that hold the data used to train and run ML models, and ultimately communicate and store model outputs for clinical use. We demonstrate through experiments with slide microscopy and computed tomography imaging, that, by bridging these two ecosystems, highdicom enables developers and researchers to train and evaluate state-of-the-art ML models in pathology and radiology while remaining compliant with the DICOM standard and interoperable with clinical systems at all stages. To promote standardization of ML research and streamline the ML model development and deployment process, we made the library available free and open-source at https://github.com/herrmannlab/highdicom .

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.

Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

Quantification of the Thoracic Aorta and Detection of Aneurysm at CT: Development and Validation of a Fully Automatic Methodology.

Purpose: To develop and validate a deep learning-based system that predicts the largest ascending and descending aortic diameters at chest CT through automatic thoracic aortic segmentation and identifies aneurysms in each segment. Materials and Methods: In this retrospective study conducted from July 2019 to February 2021, a U-Net and a postprocessing algorithm for thoracic aortic segmentation and measurement were developed by using a dataset (dataset A) that included 315 CT studies split into training, hyperparameter-tuning, and testing sets. The U-Net and postprocessing algorithm were associated with a Digital Imaging and Communications in Medicine series filter and visualization interface and were further validated by using a dataset (dataset B) that included 1400 routine CT studies. In dataset B, system-predicted measurements were compared with annotations made by two independent readers as well as radiology reports to evaluate system performance. Results: In dataset B, the mean absolute error between the automatic and reader-measured diameters was equal to or less than 0.27 cm for both the ascending aorta and the descending aorta. The intraclass correlation coefficients (ICCs) were greater than 0.80 for the ascending aorta and equal to or greater than 0.70 for the descending aorta, and the ICCs between readers were 0.91 (95% CI: 0.90, 0.92) and 0.82 (95% CI: 0.80, 0.84), respectively. Aneurysm detection accuracy was 88% (95% CI: 86, 90) and 81% (95% CI: 79, 83) compared with reader 1 and 90% (95% CI: 88, 91) and 82% (95% CI: 80, 84) compared with reader 2 for the ascending aorta and descending aorta, respectively. Conclusion: Thoracic aortic aneurysms were accurately predicted at CT by using deep learning.Keywords: Aorta, Convolutional Neural Network, Machine Learning, CT, Thorax, AneurysmsSupplemental material is available for this article.© RSNA, 2022.

Acquisition of taxane resistance by p53 inactivation in ovarian cancer cells.

Ovarian cancer is one of the most common gynecologic malignancies in women and has a poor prognosis. Taxanes are a class of standard first-line chemotherapeutic agents for the treatment of ovarian cancer. However, tumor-intrinsic and acquired resistance to taxanes poses major challenges to improving clinical outcomes. Hence, there is an urgent clinical need to understand the mechanisms of resistance in order to discover potential biomarkers and therapeutic strategies to increase taxane sensitivity in ovarian cancer. Here, we report the identification of an association between the TP53 status and taxane sensitivity in ovarian cancer cells through complementary experimental and informatics approaches. We found that TP53 inactivation is associated with taxane resistance in ovarian cancer cells, supported by the evidence from (i) drug sensitivity profiling with bioinformatic analysis of large-scale cancer therapeutic response and genomic datasets and (ii) gene signature identification based on experimental isogenic cell line models. Further, our studies revealed TP53-dependent gene expression patterns, such as overexpression of ACSM3, as potential predictive biomarkers of taxane resistance in ovarian cancer. The TP53-dependent hyperactivation of the WNT/ß-catenin pathway discovered herein revealed a potential vulnerability to exploit in developing combination therapeutic strategies. Identification of this genotype-phenotype relationship between the TP53 status and taxane sensitivity sheds light on TP53-directed patient stratification and therapeutic discoveries for ovarian cancer treatment.

Concordance between PCR-based extraction-free saliva and nasopharyngeal swabs for SARS-CoV-2 testing.

Introduction: Saliva represents a less invasive alternative to nasopharyngeal swab (NPS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. SalivaDirect is a nucleic acid extraction-free method for detecting SARS-CoV2 in saliva specimens. Studies evaluating the concordance of gold standard NPS and newly developed SalivaDirect protocols are limited. The aim of our study was to assess SalivaDirect as an alternative method for COVID-19 testing. Methods: Matching NPS and saliva samples were analysed from a cohort of symptomatic (n=127) and asymptomatic (n=181) participants recruited from hospital and university settings, respectively. RNA was extracted from NPS while saliva samples were subjected to the SalivaDirect protocol before RT-qPCR analysis. The presence of SARS-Cov-2 was assessed using RdRp and N1 gene targets in NPS and saliva, respectively. Results: Overall we observed 94.3% sensitivity (95% CI 87.2-97.5%), and 95.9% specificity (95% CI 92.4-97.8%) in saliva when compared to matching NPS samples. Analysis of concordance demonstrated 95.5% accuracy overall for the saliva test relative to NPS, and a very high level of agreement (κ coefficient = 0.889, 95% CI 0.833-0.946) between the two sets of specimens. Fourteen of 308 samples were discordant, all from symptomatic patients. Ct values were >30 in 13/14 and >35 in 6/14 samples. No significant difference was found in the Ct values of matching NPS and saliva sample ( p=0.860). A highly significant correlation (r = 0.475, p<0.0001) was also found between the Ct values of the concordant positive saliva and NPS specimens. Conclusions: Use of saliva processed according to the SalivaDirect protocol represents a valid method to detect SARS-CoV-2. Accurate and less invasive saliva screening is an attractive alternative to current testing methods based on NPS and would afford greater capacity to test asymptomatic populations especially in the context of frequent testing.