Cardiac Sarcoidosis-Diagnostic and Therapeutic Challenges.

Sarcoidosis is a multisystem disorder of unknown etiology. The leading hypothesis involves an antigen-triggered dysregulated T-cell-driven immunologic response leading to non-necrotic granulomas. In cardiac sarcoidosis (CS), the inflammatory response can lead to fibrosis, culminating in clinical manifestations such as atrioventricular block and ventricular arrhythmias. Cardiac manifestations frequently present as first and isolated signs or may appear in conjunction with extracardiac manifestations. The incidence of sudden cardiac death (SCD) is high. Diagnosis remains a challenge. For a definite diagnosis, endomyocardial biopsy (EMB) is suggested. In clinical practice, compatible findings in advanced imaging using cardiovascular magnetic resonance (CMR) and/or positron emission tomography (PET) in combination with extracardiac histological proof is considered sufficient. Management revolves around the control of myocardial inflammation by employing immunosuppression. However, data regarding efficacy are merely based on observational evidence. Prevention of SCD is of particular importance and several guidelines provide recommendations regarding device therapy. In patients with manifest CS, outcome data indicate a 5-year survival of around 90% and a 10-year survival in the range of 80%. Data for patients with silent CS are conflicting; some studies suggest an overall benign course of disease while others reported contrasting observations. Future research challenges involve better understanding of the immunologic pathogenesis of the disease for a targeted therapy, improving imaging to aid early diagnosis, assessing the need for screening of asymptomatic patients and randomized trials.

Type 1 Interferon-Stimulated Gene Expression and Disease Activity in Pediatric Rheumatic Diseases: No Composite Scores Needed?

OBJECTIVE: Rheumatic diseases are characterized by different patterns of immune overactivation. This study investigated the correlation of whole blood type 1 interferon (IFN) stimulated gene (ISG), IL18, and CXCL9 expression with clinical disease activity in pediatric rheumatic diseases and assessed the required number of ISGs to be included in a composite type 1 IFN score. METHODS: Whole blood-derived RNA and clinical data were collected from 171 mostly pediatric patients with connective tissue diseases (CTDs), systemic autoinflammatory diseases (SAIDs), monogenic interferonopathies (IFNPs) and other inflammatory diseases, and from 38 controls. The expression of six previously established ISGs, IL18, and CXCL9 was assessed by real-time polymerase chain reaction (471 samples). Individual and composite gene expression was assessed, and correlation and threshold analyses were performed. RESULTS: Correlation between ISG expression and clinical disease activity was strongest in CTD, especially in juvenile dermatomyositis (JDM) and IFNP, and modest in patients with SAID. Threshold ISG expression levels for the detection of at least mild clinical disease activity were substantially higher in patients with systemic lupus erythematosus compared with JDM. The correlation of expression levels of limited sets of ISGs and even individual ISGs with clinical disease activity were not inferior to a composite score of six ISGs. CONCLUSION: In a real-world cohort, individual ISG expression levels robustly reflected clinical disease activity in CTD and IFNP, especially in JDM, which would simplify such analyses in clinical routine and be more cost-effective. Threshold levels varied across diseases, potentially reflecting different mechanisms of type 1 IFN overactivation.

Bronchoscopist-Directed Continuous Flow Propofol Based Analgosedation during Flexible Interventional Bronchoscopy and EBUS.

Sedation techniques in interventional flexible bronchoscopy and endobronchial ultrasound-guided transbronchial-needle aspiration (EBUS-TBNA) are inconsistent and the evidence for required general anesthesia under full anesthesiologic involvement is scarce. Moreover, we faced the challenge of providing bronchoscopic care with limited personnel. Hence, we retrospectively identified 513 patients that underwent flexible interventional bronchoscopy and/or EBUS-TBNA out of our institution between January 2020 and August 2022 to evaluate our deep analgosedation approach based on pethidine/meperidine bolus plus continuous flow adjusted propofol, the bronchoscopist-directed continuous flow propofol based analgosedation (BDcfP) in a two-personnel setting. Consequently, 502 out of 513 patients received BDcfP for analgosedation. We identified cardiovascular comorbidities, chronic obstructive pulmonary disease, and arterial hypertension as risk factors for periprocedural hypotension. Propofol flow rate did not correlate with hypotension. Theodrenaline and cafedrine might be used to treat periprocedural hypotension. Moreover, midazolam might be used to support the sedative effect. In conclusion, BDcfP is a safe and feasible sedative approach during interventional flexible bronchoscopy and EBUS-TBNA. In general, after the implementation of safety measures, EBUS-TBNA and interventional flexible bronchoscopy via BDcfP might safely be performed even with limited personnel.

Risk Factors in HIV-1 Positive Patients on the Intensive Care Unit: A Single Center Experience from a Tertiary Care Hospital.

HIV-positive patients with acquired immunodeficiency syndrome (AIDS) often require treatment on intensive care units (ICUs). We aimed to present data from a German, low-incidence region cohort, and subsequently evaluate factors measured during the first 24 h of ICU stay to predict short- and long-term survival, and compare with data from high-incidence regions. We documented 62 patient courses between 2009 and 2019, treated on a non-operative ICU of a tertiary care hospital, mostly due to respiratory deterioration and co-infections. Of these, 54 patients required ventilatory support within the first 24 h with either nasal cannula/mask (n = 12), non-invasive ventilation (n = 16), or invasive ventilation (n = 26). Overall survival at day 30 was 77.4%. While ventilatory parameters (all p < 0.05), pH level (c/o 7.31, p = 0.001), and platelet count (c/o 164,000/µL, p = 0.002) were significant univariate predictors of 30-day and 60-day survival, different ICU scoring systems, such as SOFA score, APACHE II, and SAPS 2 predicted overall survival (all p < 0.001). Next to the presence or history of solid neoplasia (p = 0.026), platelet count (HR 6.7 for <164,000/µL, p = 0.020) and pH level (HR 5.8 for <7.31, p = 0.009) remained independently associated with 30-day and 60-day survival in multivariable Cox regression. However, ventilation parameters did not predict survival multivariably.

Augmented region of interest for untargeted metabolomics mass spectrometry (AriumMS) of multi-platform-based CE-MS and LC-MS data.

In mass spectrometry (MS)-based metabolomics, there is a great need to combine different analytical separation techniques to cover metabolites of different polarities and apply appropriate multi-platform data processing. Here, we introduce AriumMS (augmented region of interest for untargeted metabolomics mass spectrometry) as a reliable toolbox for multi-platform metabolomics. AriumMS offers augmented data analysis of several separation techniques utilizing a region-of-interest algorithm. To demonstrate the capabilities of AriumMS, five datasets were combined. This includes three newly developed capillary electrophoresis (CE)-Orbitrap MS methods using the recently introduced nanoCEasy CE-MS interface and two hydrophilic interaction liquid chromatography (HILIC)-Orbitrap MS methods. AriumMS provides a novel mid-level data fusion approach for multi-platform data analysis to simplify and speed up multi-platform data processing and evaluation. The key feature of AriumMS lies in the optimized data processing strategy, including parallel processing of datasets and flexible parameterization for processing of individual separation methods with different peak characteristics. As a case study, Saccharomyces cerevisiae (yeast) was treated with a growth inhibitor, and AriumMS successfully differentiated the metabolome based on the augmented multi-platform CE-MS and HILIC-MS investigation. As a result, AriumMS is proposed as a powerful tool to improve the accuracy and selectivity of metabolome analysis through the integration of several HILIC-MS/CE-MS techniques.

Radiomics of Tumor Heterogeneity in 18F-FDG-PET-CT for Predicting Response to Immune Checkpoint Inhibition in Therapy-Naïve Patients with Advanced Non-Small-Cell Lung Cancer.

We aimed to evaluate the predictive and prognostic value of baseline 18F-FDG-PET-CT (PET-CT) radiomic features (RFs) for immune checkpoint-inhibitor (CKI)-based first-line therapy in advanced non-small-cell lung cancer (NSCLC) patients. In this retrospective study 44 patients were included. Patients were treated with either CKI-monotherapy or combined CKI-based immunotherapy-chemotherapy as first-line treatment. Treatment response was assessed by the Response Evaluation Criteria in Solid Tumors (RECIST). After a median follow-up of 6.4 months patients were stratified into "responder" (n = 33) and "non-responder" (n = 11). RFs were extracted from baseline PET and CT data after segmenting PET-positive tumor volume of all lesions. A Radiomics-based model was developed based on a Radiomics signature consisting of reliable RFs that allow classification of response and overall progression using multivariate logistic regression. These RF were additionally tested for their prognostic value in all patients by applying a model-derived threshold. Two independent PET-based RFs differentiated well between responders and non-responders. For predicting response, the area under the curve (AUC) was 0.69 for "PET-Skewness" and 0.75 predicting overall progression for "PET-Median". In terms of progression-free survival analysis, patients with a lower value of PET-Skewness (threshold < 0.2014; hazard ratio (HR) 0.17, 95% CI 0.06-0.46; p < 0.001) and higher value of PET-Median (threshold > 0.5233; HR 0.23, 95% CI 0.11-0.49; p < 0.001) had a significantly lower probability of disease progression or death. Our Radiomics-based model might be able to predict response in advanced NSCLC patients treated with CKI-based first-line therapy.

High expression of transcription factor POU2F1 confers improved survival on smokers with lung adenocarcinoma: a retrospective study of two cohorts.

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide and its most important risk factor is tobacco smoking. While smoking is associated with inferior outcome in NSCLC patients, smoking also correlates with a higher tumor mutational burden. In contrast to adenocarcinomas (ADC) of non-smokers, that frequently harbor targetable gain-of-function mutations, NSCLC smokers largely present with non-targetable loss-of-function mutations of genes associated with DNA-damage repair. The transcription factor Pit-1, Oct1/2, Unc-86 (POU) domain class 2 transcription factor 1 (POU2F1) is a widely expressed bipotential stabilizer of repressed and inducible transcriptional states and frequently deregulated in cancer. Methods: Via immunohistochemistry, we evaluated POU2F1 protein expression on a tissue micro array of 217 operable stage I-III NSCLC patients. Findings were reproduced in a gene expression database of 1144 NSCLC patients, filtered for POU2F1 mRNA expression. After retroviral overexpression of POU2F1 in A549 cells, we evaluated for clonogenic growth and proliferation. Additionally, CRISPR-Cas9 mediated POU2F1 knockdown in A549 cells was likewise analyzed. Results: High protein expression of POU2F1 in 217 NSCLC patients resulted in improved outcome of smokers with ADC [hazard ratio (HR) 0.30 (0.09-0.99), P=0.035]. Moreover, gene expression analysis confirmed favorable outcome of high POU2F1 mRNA expression in smokers with ADC [HR 0.41 (0.24-0.69), P<0.001]. Other than that, retrovirally induced overexpression of POU2F1 in A549 cells significantly reduced both, clonogenic growth as well as proliferation of NSCLC cells, whereas CRISPR-Cas9 mediated knockdown of the protein did not have any impact. Conclusions: Our data suggest that high expression of POU2F1 mediates a less aggressive cancer phenotype in smokers with ADC NSCLC. Pharmacological induction of genes and signaling pathways controlled by POU2F1 may provide novel avenues for future targeted NSCLC therapies in smokers.

Enzymatic Synthesis of l-Methionine Analogues and Application in a Methyltransferase Catalysed Alkylation Cascade.

Chemical modification of small molecules is a key step for the development of pharmaceuticals. S-adenosyl-l-methionine (SAM) analogues are used by methyltransferases (MTs) to transfer alkyl, allyl and benzyl moieties chemo-, stereo- and regioselectively onto nucleophilic substrates, enabling an enzymatic way for specific derivatisation of a wide range of molecules. l-Methionine analogues are required for the synthesis of SAM analogues. Most of these are not commercially available. In nature, O-acetyl-l-homoserine sulfhydrolases (OAHS) catalyse the synthesis of l-methionine from O-acetyl-l-homoserine or l-homocysteine, and methyl mercaptan. Here, we investigated the substrate scope of ScOAHS from Saccharomyces cerevisiae for the production of l-methionine analogues from l-homocysteine and organic thiols. The promiscuous enzyme was used to synthesise nine different l-methionine analogues with modifications on the thioether residue up to a conversion of 75 %. ScOAHS was combined with an established MT dependent three-enzyme alkylation cascade, allowing transfer of in total seven moieties onto two MT substrates. For ethylation, conversion was nearly doubled with the new four-enzyme cascade, indicating a beneficial effect of the in situ production of l-methionine analogues with ScOAHS.

Biomimetic S-Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Persistent capillary rarefication in long COVID syndrome.

BACKGROUND: Recent studies have highlighted Coronavirus disease 2019 (COVID-19) as a multisystemic vascular disease. Up to 60% of the patients suffer from long-term sequelae and persistent symptoms even 6 months after the initial infection. METHODS: This prospective, observational study included 58 participants, 27 of whom were long COVID patients with persistent symptoms > 12 weeks after recovery from PCR-confirmed SARS-CoV-2 infection. Fifteen healthy volunteers and a historical cohort of critically ill COVID-19 patients (n = 16) served as controls. All participants underwent sublingual videomicroscopy using sidestream dark field imaging. A newly developed version of Glycocheck™ software was used to quantify vascular density, perfused boundary region (PBR-an inverse variable of endothelial glycocalyx dimensions), red blood cell velocity (VRBC) and the microvascular health score (MVHS™) in sublingual microvessels with diameters 4-25 µm. MEASUREMENTS AND MAIN RESULTS: Although dimensions of the glycocalyx were comparable to those of healthy controls, a µm-precise analysis showed a significant decrease of vascular density, that exclusively affected very small capillaries (D5: - 45.16%; D6: - 35.60%; D7: - 22.79%). Plotting VRBC of capillaries and feed vessels showed that the number of capillaries perfused in long COVID patients was comparable to that of critically ill COVID-19 patients and did not respond adequately to local variations of tissue metabolic demand. MVHS was markedly reduced in the long COVID cohort (healthy 3.87 vs. long COVID 2.72 points; p = 0.002). CONCLUSIONS: Our current data strongly suggest that COVID-19 leaves a persistent capillary rarefication even 18 months after infection. Whether, to what extent, and when the observed damage might be reversible remains unclear.

Substrate Induced Movement of the Metal Cofactor between Active and Resting State.

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M2+ R ), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M2+ A ) at 2.4 Šdistance. Bidentate coordination of a ketoacid substrate to M2+ A affords the overall lowest energy complex, which drives the transition from M2+ R to M2+ A . While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.

Sustained Impairment in Cardiopulmonary Exercise Capacity Testing in Patients after COVID-19: A Single Center Experience.

Background: Following COVID-19, patients often present with ongoing symptoms comparable to chronic fatigue and subjective deterioration of exercise capacity (EC), which has been recently described as postacute COVID-19 syndrome. Objective: To objectify the reduced EC after COVID-19 and to evaluate for pathologic limitations. Methods: Thirty patients with subjective limitation of EC performed cardiopulmonary exercise testing (CPET). If objectively limited in EC or deteriorated in oxygen pulse, we offered cardiac stress magnetic resonance imaging (MRI) and a follow-up CPET. Results: Eighteen male and 12 female patients were included. Limited relative EC was detected in 11/30 (36.7%) patients. Limitation correlated with reduced body weight-indexed peak oxygen (O2) uptake (peakV̇O2/kg) (mean 74.7 (±7.1) % vs. 103.6 (±14.9) %, p < 0.001). Reduced peakV̇O2/kg was found in 18/30 (60.0%) patients with limited EC. Patients with reduced EC widely presented an impaired maximum O2 pulse (75.7% (±5.6) vs. 106.8% (±13.9), p < 0.001). Abnormal gas exchange was absent in all limited EC patients. Moreover, no patient showed signs of reduced pulmonary perfusion. Using cardiac MRI, diminished biventricular ejection fraction was ruled out in 16 patients as a possible cause for reduced O2 pulse. Despite noncontrolled training exercises, follow-up CPET did not reveal any exercise improvements. Conclusions: Deterioration of EC was not associated with ventilatory or pulmonary vascular limitation. Exercise limitation was related to both reduced O2 pulse and peakV̇O2/kg, which, however, did not correlate with the initial severity of COVID-19. We hypothesize that impaired microcirculation or limited peripheral O2 utilization might be causative for prolonged deterioration of EC following acute COVID-19 infection.

High Expression of NT5DC2 Is a Negative Prognostic Marker in Pulmonary Adenocarcinoma.

Via immunohistochemistry (IHC) on tissue micro arrays (TMA) clinical and prognostic impact of p53 co-playing 5'-Nucleotidase Domain-Containing Protein 2 (NT5DC2) protein expression was evaluated in 252 NSCLC patients. Confirmatory, gene expression database. mRNA levels of NT5DC2 were studied in 1925 NSCLC patients. High protein expression of NT5DC2 resulted in reduced median overall survival (OS) of patients with stage I-III adenocarcinoma (ADC) (Log Rank p = 0.026, HR 2.04 (1.08−3.87)), but not in squamous cell carcinoma (SCC) (p = 0.514, HR 0.87 (0.57−1.33)). Findings on OS were reproduced via gene expression analysis in ADC (p < 0.001, HR 1.64 (1.30−2.08)) and SCC (p = 0.217, HR 0.86 (0.68−1.09)). Yet, NT5DC2 mRNA levels were higher in SCC compared to ADC (p < 0.001) and in pN2 tumors compared to pN0/1 tumors (p = 0.001). Likewise, NT5DC2 protein expression associated with high-grade SCC. Moreover, NT5DC2 expression was positively correlated with p53 protein (p = 0.018) and TP53 gene expression (p < 0.001) and its survival effect was p53 dependent. While p53 expression was negatively associated with the presence of CD34+ cancer associated fibroblasts (CAFs), NT5DC2 expression insignificantly tended to higher levels of SMA+ CAFs (p = 0.065).

Central airway obstruction treatment with self-expanding covered Y-carina nitinol stents: A single center retrospective analysis.

BACKGROUND: Central airway obstruction (CAO) is one of the most challenging, potentially lethal complications in malignant and benign respiratory diseases. Worsening dyspnea is also a relevant cause for reduced quality of life in such patients. Here, we present our data on the application of covered, self-expanding Y-carina nitinol stents due to benign and malignant diseases. METHODS: We retrospectively identified 27 patients who had undergone 31 rigid bronchoscopies with implantation of covered Y-carina nitinol stents over a period of 10 years in order to evaluate indication, clinical course, and outcome. RESULTS: Short-term survival of successfully stented patients with palliative and curative treatment goal did not differ, allowing for diagnosis independent indication. With respect to overall survival, patients with endoluminal obstruction benefited most compared to patients with fistula and/or external compression. Granulation tissue formation (61.3%) and mucus plugging (80.6%) were the most frequent complications. Material defect (6.5%) and migration (3.2%) were rare complications that could be handled by revisional rigid bronchoscopy and stent exchange in some cases. CONCLUSIONS: Implantation of self-expanding covered Y-carina nitinol stents via rigid bronchoscopy is a feasible and safe treatment option for benign and malignant central airway obstruction. Especially in palliative, malignant airway stenosis, stenting might facilitate additional treatment options and optimize dyspnea and eventually quality of life.

The Diagnostic Arthroscopy Skill Score (DASS): a reliable and suitable assessment tool for arthroscopic skill training.

PURPOSE: To develop and validate a novel score to more objectively assess the performance of diagnostic knee arthroscopy using a simulator. METHODS: A Diagnostic Arthroscopy Skill Score (DASS) was developed by ten AGA (AGA-Society for Arthroscopy and Joint-Surgery) instructors for the assessment of arthroscopic skills. DASS consists of two parts: the evaluation of standardized diagnostic knee arthroscopy (DASSpart1) and the evaluation of manual dexterity, including ambidexterity and triangulation, using objective measurement parameters (DASSpart2). Content validity was determined by the Delphi method. One hundred and eleven videos of diagnostic knee arthroscopies were recorded during simulator training courses and evaluated by six specially trained instructors using DASS. Construct validity, measurement error calculated by the minimum detectable change (MDC), internal consistency using Cronbach's alpha and interrater and intrarater reliability were assessed. The Bland-Altman method was used to calculate the intrarater agreement. RESULTS: Six skill domains were identified and evaluated for each knee compartment. DASS, DASSpart1, and DASSpart2 showed construct validity, with experts achieving significantly higher scores than competents and novices. MDC was 4.5 ± 1.7 points for DASSpart1. There was high internal consistency for all domains in each compartment from 0.78 to 0.86. The interrater reliability showed high agreement between the six raters (ICC = 0.94). The evaluation of intrarater reliability demonstrated good and excellent agreement for five raters (ICC > 0.80) and moderate agreement for one rater (ICC = 0.68). The Bland-Altman comparison showed no difference between the first and second evaluations in five out of six raters. Precision, estimated by the regression analysis and comparison with the method of Bland and Altman, was excellent for four raters and moderate for two raters. CONCLUSIONS: The results of this study indicate good validity and reliability of DASS for the assessment of the surgical performance of diagnostic knee arthroscopy during simulator training. Standardized training is recommended before arthroscopy surgery is considered in patients. LEVEL OF EVIDENCE: II.

Ten hours of simulator training in arthroscopy are insufficient to reach the target level based on the Diagnostic Arthroscopic Skill Score.

PURPOSE: Simulator arthroscopy training has gained popularity in recent years. However, it remains unclear what level of competency surgeons may achieve in what time frame using virtual training. It was hypothesized that 10 h of training would be sufficient to reach the target level defined by experts based on the Diagnostic Arthroscopic Skill Score (DASS). METHODS: The training concept was developed by ten instructors affiliated with the German-speaking Society of Arthroscopy and Joint Surgery (AGA). The programme teaches the basics of performing arthroscopy; the main focus is on learning and practicing manual skills using a simulator. The training was based on a structured programme of exercises designed to help users reach defined learning goals. Initially, camera posture, horizon adjustment and control of the direction of view were taught in a virtual room. Based on these skills, further training was performed with a knee model. The learning progress was assessed by quantifying the exercise time, camera path length and instrument path length for selected tasks. At the end of the course, the learners' performance in diagnostic arthroscopy was evaluated using DASS. Participants were classified as novice or competent based on the number of arthroscopies performed prior to the assessment. RESULTS: Except for one surgeon, 131 orthopaedic residents and surgeons (29 women, 102 men) who participated in the seven courses agreed to anonymous data analysis. Fifty-eight of them were competents with more than ten independently performed arthroscopies, and 73 were novices, with fewer than ten independently performed arthroscopies. There were significant reductions in exercise time, camera path length and instrument path length for all participants after the training, indicating a rapid increase in performance. No difference in camera handling between the dominant and non-dominant sides was found in either group. The competents performed better than the novices in various tasks and achieved significantly better DASS values on the final performance test. CONCLUSIONS: Our data have demonstrated that arthroscopic skills can be taught effectively on a simulator, but a 10-h course is not sufficient to reach the target level set by experienced arthroscopists. However, learning progress can be monitored more objectively during simulator training than in the operating room, and simulation may partially replace the current practice of arthroscopic training. LEVEL OF EVIDENCE: III.

A cardiovascular magnetic resonance imaging-based pilot study to assess coronary microvascular disease in COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and is primarily characterised by a respiratory disease. However, SARS-CoV-2 can directly infect vascular endothelium and subsequently cause vascular inflammation, atherosclerotic plaque instability and thereby result in both endothelial dysfunction and myocardial inflammation/infarction. Interestingly, up to 50% of patients suffer from persistent exercise dyspnoea and a post-viral fatigue syndrome (PVFS) after having overcome an acute COVID-19 infection. In the present study, we assessed the presence of coronary microvascular disease (CMD) by cardiovascular magnetic resonance (CMR) in post-COVID-19 patients still suffering from exercise dyspnoea and PVFS. N = 22 patients who recently recovered from COVID-19, N = 16 patients with classic hypertrophic cardiomyopathy (HCM) and N = 17 healthy control patients without relevant cardiac disease underwent dedicated vasodilator-stress CMR studies on a 1.5-T MR scanner. The CMR protocol comprised cine and late-gadolinium-enhancement (LGE) imaging as well as velocity-encoded (VENC) phase-contrast imaging of the coronary sinus flow (CSF) at rest and during pharmacological stress (maximal vasodilation induced by 400 µg IV regadenoson). Using CSF measurements at rest and during stress, global myocardial perfusion reserve (MPR) was calculated. There was no difference in left ventricular ejection-fraction (LV-EF) between COVID-19 patients and controls (60% [57-63%] vs. 63% [60-66%], p = NS). There were only N = 4 COVID-19 patients (18%) showing a non-ischemic pattern of LGE. VENC-based flow measurements showed that CSF at rest was higher in COVID-19 patients compared to controls (1.78 ml/min [1.19-2.23 ml/min] vs. 1.14 ml/min [0.91-1.32 ml/min], p = 0.048). In contrast, CSF during stress was lower in COVID-19 patients compared to controls (3.33 ml/min [2.76-4.20 ml/min] vs. 5.32 ml/min [3.66-5.52 ml/min], p = 0.05). A significantly reduced MPR was calculated in COVID-19 patients compared to healthy controls (2.73 [2.10-4.15-11] vs. 4.82 [3.70-6.68], p = 0.005). No significant differences regarding MPR were detected between COVID-19 patients and HCM patients. In post-COVID-19 patients with persistent exertional dyspnoea and PVFS, a significantly reduced MPR suggestive of CMD-similar to HCM patients-was observed in the present study. A reduction in MPR can be caused by preceding SARS-CoV-2-associated direct as well as secondary triggered mechanisms leading to diffuse CMD, and may explain ongoing symptoms of exercise dyspnoea and PVFS in some patients after COVID-19 infection.

The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family.

CYTH proteins make up a large superfamily that is conserved in all three domains of life. These enzymes have a triphosphate tunnel metalloenzyme (TTM) fold, which typically results in phosphatase functions, e.g., RNA triphosphatase, inorganic polyphosphatase, or thiamine triphosphatase. Some CYTH orthologs cyclize nucleotide triphosphates to 3',5'-cyclic nucleotides. So far, archaeal CYTH proteins have been annotated as adenylyl cyclases, although experimental evidence to support these annotations is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our in silico studies derived ten major subclasses within the CYTH family implying a close relationship between these archaeal CYTH enzymes and class IV adenylyl cyclases. However, initial biochemical characterization reveals inability of SaTTM to produce any cyclic nucleotides. Instead, our structural and functional analyses show a classical TTM behavior, i.e., triphosphatase activity, where pyrophosphate causes product inhibition. The Ca2+-inhibited Michaelis complex indicates a two-metal-ion reaction mechanism analogous to other TTMs. Cocrystal structures of SaTTM further reveal conformational dynamics in SaTTM that suggest feedback inhibition in TTMs due to tunnel closure in the product state. These structural insights combined with further sequence similarity network-based in silico analyses provide a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.