Aldehyde-mediated inhibition of asparagine biosynthesis has implications for diabetes and alcoholism.

Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.

Rising use of diagnostic imaging in Australia: An analysis of Medicare-funded radiology services between 2000 and 2021.

INTRODUCTION: The use of diagnostic imaging services is increasing worldwide. This has important impacts on healthcare resource allocation and potential risks to the population. This study aimed to quantify trends in medical imaging in Australia over the past two decades. METHODS: Data were extracted from the Australian Medicare Benefits Schedule (MBS) between 2000 and 2021. Simple linear regression analyses were performed to assess changes in absolute utilisation and utilisation rate per 100,000 population of total imaging services as well as by each imaging modality. Logistic regression analysis was performed to assess changes in total imaging services as a proportion of total Medicare services over time. Chi-squared test was used to assess for change in modality composition of imaging services. RESULTS: There were 436,255,500 imaging studies performed between 2000 and 2021. The absolute utilisation of total imaging services increased annually by an average of 864,404 (95% CI: 808,235-920,573, p < 0.001). For each consecutive year, the proportion of total Medicare services attributed to total imaging services increased by 0.01% (95% CI: 0.01-0.01, p < 0.01). There was also a statistically significant increase in the utilisation rates of imaging services per 100,000 population for each imaging modality. The number of imaging services per radiologist increased on average by 74 (95% CI: 26-122, p < 0.05) annually. CONCLUSION: The utilisation of diagnostic imaging services has increased in Australia between 2000 and 2021, outpacing the population growth, total healthcare services, and the radiologist workforce.

Formaldehyde reacts with N-terminal proline residues to give bicyclic aminals.

Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.

The impact of the COVID-19 pandemic on diagnostic imaging services in Australia.

INTRODUCTION: The COVID-19 pandemic is driving unprecedented changes in healthcare services worldwide. This study aimed to quantify the impact of the first wave of the COVID-19 pandemic on diagnostic imaging services in Australia using an interrupted time series model. METHODS: Monthly data were extracted from the Australian Medicare Benefits Schedule for all diagnostic imaging services performed between January 2016 and December 2019. Holt-Winters forecasting models were developed for total imaging services as well as for each imaging modality. The models were used to predict monthly data between January 2020 and June 2020 with a 95% confidence interval (P < 0.05). Absolute and percentage residual differences (RD) between observed and predicted services for this time period were calculated. RESULTS: There were statistically significant reductions in total imaging services performed in March 2020 (RD: -332260, -13.1%, 95% CI: -17.5% to -8.4%), April 2020 (RD: -716957, -32.4%, 95% CI: -36.2% to -28.1%) and May 2020 (RD: -571634, -21.4%, 95% CI: -25.1% to -17.3%). Nuclear medicine and CT services were relatively less impacted than general radiography, ultrasound, and MRI services. There was also a statistically significant increase in nuclear medicine and CT services performed in June 2020 compared to predicted values. CONCLUSIONS: During the first wave of COVID-19 in Australia, there was a significant reduction in total diagnostic imaging services, with variable impacts on different imaging modalities. These findings may have significant public health implications and can be used to inform evidence-based strategies in the recovery phase of the pandemic.

Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding.

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective 'peptibitors' inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.

Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1.

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a 'gain-of-function' to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

Mass spectrometry reveals potential of ß-lactams as SARS-CoV-2 Mpro inhibitors.

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.

Metampicillin is a cyclic aminal produced by reaction of ampicillin with formaldehyde.

Metampicillin is a ß-lactam antibiotic that is prepared by the reaction of ampicillin with formaldehyde. Although metampicillin has been studied for treatment of infections in animals and humans, its structure has been unclear. We report NMR studies revealing that metampicillin contains a formaldehyde-derived cyclic aminal. NMR time-course experiments with excess formaldehyde in solution show formation of another product with an additional exocyclic hemiaminal group formed by reaction with the cyclic aminal nitrogen. The exocyclic hemiaminal group is readily removed by reaction with the formaldehyde scavenger 1,3-cyclohexanedione, whereas the cyclic aminal methylene exhibits greater stability. The overall results assign the structure of metampicillin as containing a cyclic aminal and further reveal the potential for complexity in the reaction of formaldehyde with biomedicinally relevant molecules.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

Opercular perivascular cysts: A proposed new subtype of dilated perivascular spaces.

PURPOSE: Dilated perivascular spaces are a common finding on brain MRI, traditionally classified into three types based on location and relationship to vessels. Recent studies have characterised an additional variant of dilated perivascular spaces that arise within the anterior temporal lobe and have unique neuroimaging features. These particular perivascular spaces are associated with a vascular loop of a branch of the middle cerebral artery (MCA) and commonly demonstrate perilesional T2/FLAIR signal. To our knowledge, these have not previously been described in the frontal lobe. METHOD: Dilated perivascular spaces associated with a vascular loop of a branch of the middle cerebral artery (MCA) identified at our institution were reviewed for imaging characteristics and anatomical location. RESULTS: 18 cases were identified. 16 were located in the anterior temporal lobe and two were located in the frontal operculum. All demonstrated internal signal characteristics identical to CSF on all sequences, with no contrast enhancement or susceptibility artefact and variable perilesional T2/FLAIR signal. CONCLUSIONS: We report further evidence of a distinct subtype of dilated perivascular spaces occurring in the anterior temporal lobe in association with a vascular loop of a branch of the MCA. In addition, we have demonstrated that these may also occur in the frontal operculum. We therefore suggest that these dilated perivascular spaces of the operculum be recognised as a separate, fourth, subtype of perivascular space and propose the term "opercular perivascular cyst".

Pernicious azotaemia? A case series of subacute combined degeneration of the cord secondary to nitrous oxide abuse.

Nitrous oxide abuse is a rare cause of vitamin B12 deficiency and consequent subacute combined degeneration of the spinal cord. Worldwide and Australian statistics indicate that recreational use of nitrous oxide is increasing. We report four cases of females aged 18-24 years presenting with clinical symptoms of subacute combined degeneration of the spinal cord. MRI during admission demonstrated the classic findings of T2 hyperintensity, predominantly within the dorsal columns of the spinal cord, with variable involvement of the lateral corticospinal tracts. These cases highlight the ready availability of nitrous oxide and the fact that heavy prolonged recreational use is occurring in the community. It is important that clinicians in emergency and community settings are alerted to this unusual cause of subacute combined degeneration of the spinal cord because early aggressive vitamin B12 replacement together with behavioural change can reverse this disabling neurological syndrome.

Formaldehyde quantification using ampicillin is not selective.

Formaldehyde (HCHO) is a simple and highly reactive human metabolite but its biochemistry is poorly defined. A limiting factor in HCHO research is lack of validated quantification methods for HCHO relevant to biological samples. We describe spectroscopic studies on a reported fluorescence-based HCHO detection method involving its reaction with ampicillin. The results validate the structure and fluorescence properties of the HCHO-ampicillin reaction product. However, the same adduct is observed after reaction of ampicillin with glyoxylate. Related fluorophores were formed with other biologically relevant carbonyl compounds. Overall, our studies suggest the ampicillin method is not reliable for selective detection and quantification of HCHO in biological samples.

Extraosseous Ewing sarcoma arising in a chronically lymphedematous limb.

Ewing sarcoma is a highly aggressive small round cell malignant neoplasm of bone and soft tissue that typically is manifested in children and young adults. It is most commonly a primary bone tumor; however, extraosseous cases have been increasingly reported. We report a case of metastatic extraosseous Ewing sarcoma with the primary lesion occurring within a limb affected by primary lymphedema. Lymphedema, in this case, played a role not only in the genesis of the tumor because of the relative local immunosuppression but also in masking the development of the lower limb mass.

Rationale and design of the ICON-RELOADED study: International Collaborative of N-terminal pro-B-type Natriuretic Peptide Re-evaluation of Acute Diagnostic Cut-Offs in the Emergency Department.

OBJECTIVES: The objectives were to reassess use of amino-terminal pro B-type natriuretic peptide (NT-proBNP) concentrations for diagnosis and prognosis of acute heart failure (HF) in patients with acute dyspnea. BACKGROUND: NT-proBNP facilitates diagnosis, prognosis, and treatment in patients with suspected or proven acute HF. As demographics of such patients are changing, previous diagnostic NT-proBNP thresholds may need updating. Additionally, value of in-hospital NT-proBNP prognostic monitoring for HF is less understood. METHODS: In a prospective, multicenter study in the United States and Canada, patients presenting to emergency departments with acute dyspnea were enrolled, with demographic, medication, imaging, and clinical course information collected. NT-proBNP analysis will be performed using the Roche Diagnostics Elecsys proBNPII immunoassay in blood samples obtained at baseline and at discharge (if hospitalized). Primary end points include positive predictive value of previously established age-stratified NT-proBNP thresholds for the adjudicated diagnosis of acute HF and its negative predictive value to exclude acute HF. Secondary end points include sensitivity, specificity, and positive and negative likelihood ratios for acute HF and, among those with HF, the prognostic value of baseline and predischarge NT-proBNP for adjudicated clinical end points (including all-cause death and hospitalization) at 30 and 180days. RESULTS: A total of 1,461 dyspneic subjects have been enrolled and are eligible for analysis. Follow-up for clinical outcome is ongoing. CONCLUSIONS: The International Collaborative of N-terminal pro-B-type Natriuretic Peptide Re-evaluation of Acute Diagnostic Cut-Offs in the Emergency Department study offers a contemporary opportunity to understand best diagnostic cutoff points for NT-proBNP in acute HF and validate in-hospital monitoring of HF using NT-proBNP.

Rare "Inverted" Verrucous Carcinoma (Carcinoma Cuniculatum) of the Sacrogluteal Region: Case Report and Literature Review.

A 49-year-old woman with a past history of several infundibular cysts in the head and neck region presented with a 6-month history of a nonhealing perianal abscess on a background of 13 years of chronic sacral-buttock abscesses and discharging sinuses as part of hidradenitis suppurativa. After 2 surgical procedures and inadequate healing, a wide local excision was performed. The surgical specimen revealed an extensive, infiltrating, cystic and deeply penetrating, dermal and subcutaneous neoplasm with multiple fistulous tracts extending to the skin surface. The histopathology was consistent with carcinoma cuniculatum, a rare, slow growing, verucciform variant of squamous cell carcinoma. An entirely subcutaneous verrucous carcinoma of the sacrogluteal region is exceptionally rare, and this represents the first documentation of such a case in a female.

The new and old definitions of acute myocardial infarction: a data-based comparison.

PURPOSE: To measure the agreement between the newer European Society of Cardiology-American College of Cardiology (ESC-ACC) definition of acute myocardial infarction (AMI) and the traditional definition established by the World Health Organization (WHO). BASIC PROCEDURES: All adult ED patients admitted to our institution with at least one abnormally elevated cardiac biomarker were determined to have had an AMI by either or both definitions. The degree of agreement and the frequency of the reasons for disagreement between these 2 definitions were measured. MAIN FINDINGS: A final study population consisted of 339 patients; 196 (58%) had an AMI by one or both definitions. Among them, 126 (64%; 95% confidence interval [CI], 57-71) were discordant for these 2 definitions: 104 (53%; 95% CI, 46-60) met only the ESC-ACC, whereas 22 (11%; 95% CI, 6-16) met only the WHO definition. Among those who met only the ESC-ACC definition, 37 (36%; 95% CI, 27-45) met none of the 3 traditional WHO criteria. PRINCIPAL CONCLUSIONS: More patients are discordant than concordant for the 2 standard definitions of AMI. Among them, a large majority meet only the new ESC-ACC definition.