TMPRSS2 isoform 1 downregulation by G-quadruplex stabilization induces SARS-CoV-2 replication arrest.

BACKGROUND: SARS-CoV-2 infection depends on the host cell factors angiotensin-converting enzyme 2, ACE2, and the transmembrane serinprotease 2, TMPRSS2. Potential inhibitors of these proteins would be ideal targets against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection. Our data opens the possibility that changes within TMPRSS2 can modulate the outcome during a SARS-CoV-2 infection. RESULTS: We reveal that TMPRSS2 acts not only during viral entry but has also an important role during viral replication. In addition to previous functions for TMPRSS2 during viral entry, we determined by specific downregulation of distinct isoforms that only isoform 1 controls and supports viral replication. G-quadruplex (G4) stabilization by chemical compounds impacts TMPRSS2 gene expression. Here we extend and in-depth characterize these observations and identify that a specific G4 in the first exon of the TMPRSS2 isoform 1 is particular targeted by the G4 ligand and affects viral replication. Analysis of potential single nucleotide polymorphisms (SNPs) reveals that a reported SNP at this G4 in isoform 1 destroys the G4 motif and makes TMPRSS2 ineffective towards G4 treatment. CONCLUSION: These findings uncover a novel mechanism in which G4 stabilization impacts SARS-CoV-2 replication by changing TMPRSS2 isoform 1 gene expression.

Diagnostic Ultrasound for Acute Appendicitis: The Gold Standard.

BACKGROUND: Acute appendicitis may present a diagnostic dilemma. The aim of this study was to review the accuracy of ultrasound in the diagnosis of paediatric acute appendicitis. METHOD: Ultrasound studies performed for investigation of appendicitis during 2015-2021 were retrieved from a tertiary paediatric hospital database and reviewed. Medical records were reviewed to determine operative intervention, further imaging, and final diagnosis. Diagnostic accuracy was assessed by sensitivity, specificity, predictivity, and overall accuracy. All appendicectomy specimens underwent histopathological confirmation. This study was approved by the local Human Research Ethics Committee. RESULTS: A total of 8555 consecutive ultrasound examinations were performed during the study period. Mean patient age was 10.8 years ( ± 3.7). Overall diagnostic accuracy was 96.1% (8221/8555) with a visualisation rate of 91.0%. Sensitivity and specificity were 96.2% (CI 95.3-97.0%) and 96.1% (CI 95.6-96.5%), respectively. When limited to positive/negative scans, sensitivity was 99.6% (CI 99.2-99.8%) and specificity 99.0% (CI 98.7-99.3%). Positive and negative predictive values were 96.9% and 99.9%, respectively. Repeat ultrasound following a non-diagnostic scan led to a definitive diagnosis in 76.1%. Negative appendicectomy rate was 5.5% overall in children who had undergone pre-operative ultrasound (107/1938), and 4.4% when other surgical pathologies were excluded. CONCLUSION: Ultrasound examination provides gold-standard accuracy in the diagnosis of paediatric appendicitis and reduces rates of negative appendicectomy. Given the disadvantages of computed tomography and magnetic resonance imaging, ultrasound should be considered the first-line investigation of choice in the diagnosis of acute appendicitis in children. LEVEL OF EVIDENCE: III.

The Global Characterisation of a Drug-Dendrimer Conjugate - PEGylated poly-lysine Dendrimer.

The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.

The Relevance of G-Quadruplexes for DNA Repair.

DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.

Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Benchmarking the value of ultrasound for acute appendicitis in children.

BACKGROUND: This study appraises the diagnostic quality of ultrasound for acute appendicitis in children and consequently challenges the perception of inferior accuracy and suitability compared to computed tomography (CT). METHODS: Radiologist reports for consecutive "query appendicitis" ultrasound studies were retrieved from a hospital database for the study period 2009-2014. Children who subsequently underwent appendicectomy were identified. Corresponding operative and histopathology findings were evaluated. Diagnostic accuracy of ultrasound was determined by analyzing overall accuracy, sensitivity, specificity, predictivity, and likelihood ratios. RESULTS: A total of 3799 ultrasound examinations were evaluated. Mean age was 11.5±3.8years. The proportion of patients investigated with preoperative ultrasound was 59.9% (1103/1840). Appendix visualization rate was 91.7%. Overall diagnostic accuracy was 95.5%. Sensitivity and specificity values were 97.1% (95.9-98.1; 95% CI) and 94.8% (93.9-95.6; 95% CI), respectively. Separate analysis of only ultrasound positive and negative examinations (i.e., excluding nondiagnostic examinations) confirmed sensitivity and specificity values of 98.8% and 98.3%. CONCLUSION: In this largest reported single institution series of ultrasound examinations for appendicitis, we report benchmark standard quality of diagnostic accuracy and visualization rates. Given the radiation and cost implications of CT, there is a strong argument to recommend ultrasound as the primary imaging modality. Diagnostic Study-Level II.

A clinical audit of anatomical side marker use in a paediatric medical imaging department.

INTRODUCTION: The gold standard in general radiography is to place a radiopaque anatomical side marker in the field of view for each radiographic image prior to exposure. The advent of digital radiography has allowed for anatomical side markers to be digitally added to films as part of post-processing. The aim of this audit was to identify whether general X-ray images performed in a tertiary Women's and Children's Hospital were being appropriately annotated with a definitive side marker, and to identify factors that may contribute to inappropriately labelled images. METHODS: Four hundred images from 201 patients' examinations occurring within a randomly selected time period were assessed to ascertain whether radiographic anatomical side markers were visible when images were viewed via the hospitals main viewing platform. The audit occurred in January 2014. The scope included both mobile and in-department general X-ray examinations, with the patient age range extending from 1 day to 18 years. RESULTS: Of the 400 images evaluated, 88 (22%) were found to have a lead marker that matched the anatomy being imaged within the primary beam; 289 (72.3%) images contained a correct digital marker inserted as part of the post-processing of the image. In total, 377 (94.2%) images were appropriately marked. Of the 23 (5.8%) images not marked correctly, 22 images had no marker and 1 was incorrectly marked with a digital marker. There was a noticeable relationship between absent anatomical markers and chest X-rays performed outside of the medical imaging department. CONCLUSIONS: While it is encouraging that the majority of the images assessed were correctly annotated, with only a small number of missing markers, there are opportunities for further improvement. The audit findings suggest that reduced access to lead markers influences marker use. Strategies that may improve compliance at an individual level include distribution of personalised anatomical side markers, and targeted staff education sessions. At a department level, regular audits and monitoring should be encouraged.

Diagnostic reference levels of paediatric computed tomography examinations performed at a dedicated Australian paediatric hospital.

INTRODUCTION: Diagnostic Reference Levels (DRL) of procedures involving ionizing radiation are important tools to optimizing radiation doses delivered to patients and in identifying cases where the levels of doses are unusually high. This is particularly important for paediatric patients undergoing computed tomography (CT) examinations as these examinations are associated with relatively high-dose. METHODS: Paediatric CT studies, performed at our institution from January 2010 to March 2014, have been retrospectively analysed to determine the 75th and 95th percentiles of both the volume computed tomography dose index (CTDIvol ) and dose-length product (DLP) for the most commonly performed studies to: establish local diagnostic reference levels for paediatric computed tomography examinations performed at our institution, benchmark our DRL with national and international published paediatric values, and determine the compliance of CT radiographer with established protocols. RESULTS: The derived local 75th percentile DRL have been found to be acceptable when compared with those published by the Australian National Radiation Dose Register and two national children's hospitals, and at the international level with the National Reference Doses for the UK. The 95th percentiles of CTDIvol for the various CT examinations have been found to be acceptable values for the CT scanner Dose-Check Notification. Benchmarking CT radiographers shows that they follow the set protocols for the various examinations without significant variations in the machine setting factors. CONCLUSION: The derivation of DRL has given us the tool to evaluate and improve the performance of our CT service by improved compliance and a reduction in radiation dose to our paediatric patients. We have also been able to benchmark our performance with similar national and international institutions.

Diagnostic reference levels for common paediatric fluoroscopic examinations performed at a dedicated paediatric Australian hospital.

INTRODUCTION: Diagnostic reference levels (DRL) of procedures involving ionising radiation are important tools for optimising radiation doses delivered to patients and to identify cases where the levels of dose are unusually high. This is particularly important for paediatric patients undergoing fluoroscopic examinations as these examinations can be associated with a high radiation dose. In this study, a large amount of paediatric fluoroscopic data has been analysed to: establish local DRL, identify the most significant factors determining radiation dose to patients, and modify fluoroscopic techniques to optimise the examination protocols. METHODS: Paediatric fluoroscopic studies performed at our institution from April 2010 to May 2015 have been retrospectively analysed to determine range, mean, 75th and 95th percentiles of Dose-Area Product (DAP) and fluoroscopic screening time for Micturating Cystourethrography (MCU), Airway, Airway and Swallow, Barium Swallow and Meal, Barium Follow Through and Barium Enema studies. RESULTS: Currently, no Australian paediatric fluoroscopic DRL data are available for comparison and thus our data can only be compared with international published data. No major changes to examination protocols or modification to fluoroscopic techniques were found necessary as our data compared well with the international published values. CONCLUSION: The dose delivered to patients depend on a number of factors particularly the experience of the operators. However, DRL are also important, as shown in this study, as they enable best practice by providing feedback to the operators on their performance and benchmarking the institution with other institutions.

Detection of biofilm formation and nanobacteria under long-term cell culture conditions in serum samples of cattle, goats, cats, and dogs.

OBJECTIVE: To determine the prevalence of biofilm formation under long-term cell culture conditions in serum samples of dairy cattle, goats, cats, and dogs, and to determine whether there is an association between nanobacteria and biofilm formation. SAMPLE POPULATION: Serum samples of clinically normal animals (313 dairy cattle, 48 goats, 140 dogs, and 44 cats) and animals with various medical conditions (60 dogs and 116 cats). PROCEDURE: Serum was incubated under cell culture conditions and observed for biofilm formation by use of light microscopy, electron microscopy, and spectroscopy. A polymerase chain reaction assay was developed to identify 16S rRNA gene sequences of nanobacteria. RESULTS: Biofilm formation developed in serum samples of 304 of 313 (97%) cattle, 44 of 48 (92%) goats, 44 of 44 (100%) cats, and 126 of 140 (90%) dogs. Prevalence of serum samples with positive results for biofilm formation was not significantly different between cats or dogs with and without medical conditions associated with pathologic extraskeletal calcification processes. Scanning electron microscopy and spectroscopy of biofilm samples revealed small coccoid particles consisting mainly of calcium and phosphate. Polymerase chain reaction assay failed to amplify sequences of nanobacteria. CONCLUSIONS AND CLINICAL RELEVANCE: Under long-term cell culture conditions, biofilm made up of aggregates of calcium and phosphate crystals does form in serum samples of clinically normal dairy cattle, goats, cats, and dogs. Disease, however, does not predispose to biofilm formation in serum samples of dogs and cats. Our findings did not support the existence of nanobacteria in serum samples of cattle, goats, cats, and dogs.