Characterization of Enzymes Involved in the Synthesis and Processing of Ether Lipids in Archaea and Bacteria

In Archaea, ether lipids play an essential role as the main building blocks of the cellular membrane. Recently, ether lipids have also been discovered in the domain of Bacteria, and the key enzymes that catalyze their synthesis, glycerol 1-phosphate dehydrogenase (AraM) and heptaprenylglyceryl phosphate synthase (HepGPS), have been described. In Bacillales, heptaprenylglyceryl phosphate (HepGP) does not become linked to a second polyprenyl moiety like for ether lipids in Archaea, but is dephosphorylated and acetylated. The enzymes that catalyze these reactions have been identified and characterized whithin this work. The phosphatase PhoB acting on HepGP was enriched from a Bacillus subtilis (B. subtilis) cell extract and identified by mass spectrometry. The B. subtilis gene phoB was amplified and heterologously expressed. The dephosphorylation activity of PhoB could be verified in vitro. Nevertheless, the results indicate that any other phosphatase might catalyze dephosphorylation of HepGP as well, because those enzymes only exhibit a low subtrate specificity. By screening a B. subtilis knockout library for deficiency in acetylation, the yvoF gene product was identified to be the acetyltransferase. The yvoF gene was heterologously expressed and YvoF was characterized biochemically. Its acetyl-CoA dependent activity was verified in vitro and catalytic parameters were obtained by a 5,5´-dithiobis-(2-nitrobenzoic acid)-coupled activity assay and a discontinuous radiometric assay. A membrane assoziation of YvoF was inferred from ultracentrifugation experiments. The in vitro acetyltransferase activity was also verified for another YvoF representative, namely from Bacillus anthracis. Because YvoF is a close relative to maltose O-acetyltransferase (MAT), the activity of YvoF was compared to MAT. YvoF and MAT partially overlap in substrate and product range in vitro, but MAT is not able to complement the yvoF knockout in vivo. It remains to be clarified how this in vivo specialization is achieved, and one answer might be that the cellular localization of the two enzymes is different, as the YvoF substrate HepG is associated to the cellular membrane. The biological function of acetylated ether lipids in B. subtilis is still unknown. However, the identification of two enzymes involved in their biosynthesis pathway brings us closer to answer this intriguing question. The HepGPS belongs to the family of geranylgeranylglyceryl phosphate synthases (GGGPS) and previous studies showed that the members of this family can be dimers or hexamers. Hexameric GGGPS complexes are built from three dimeric modules in the configuration of the native dimers. Besides the dimeric interface (symmetric interface 1), two additional interfaces exist in the hexamer, the symmetric interface 2 and the asymmetric interface 3a/3b. To elucidate the impact of hexamerization on stability and activity, the GGGPS from Methanothermobacter thermautotrophicus (mtGGGPS) was investigated by mutational studies. The symmetric interface 2 and the asymmetric interface 3a/3b were shown to contribute equally to the stabilization of the hexameric structure, because both led to dimerization if disturbed. A mutation that disrupts the dimeric interface led to a further collapse of the dimers into monomers. The thermal stability of the resulting monomeric, dimeric and hexameric mtGGGPS mutants was determined via various biophysical techniques (circular dichroism-, differential scanning calorimetry-, nano differential scanning fluorimetry-measurements and irreversible heat inactivation). In almost all cases, a higher oligomerization state ensured an elevated thermostability and contributed to maintaining activity at higher temperatures. Based on these results, a putative unfolding pathway for mtGGGPS was proposed. First, a transition of the native structure to a stable partially folded intermediate occurs, which is inactive but maintains almost 80-90 % of the overall secondary structure. Depending on the oligomerization state, this 1st transition happens at a temperature between 50 °C (monomeric mutants), 60 °C (dimeric mutants) and 100 °C (hexameric wild type and mutants). Only at elevated temperatures of around 120 °C a second transition presumably leads to a complete denaturation of the protein, irrespective of the oligomerization state. Data obtained with two other couples of hexameric GGGPS wild type and dimeric mutant as well as the analysis of native hexameric and dimeric GGGPS enzymes from different species confirmed the results obtained with mtGGGPS. A steady-state kinetic experiment revealed that the dimeric mutants showed decreased catalytic efficiencies especially due to an increase of the KM for G1P. In summary, it could be shown for almost all hexameric enzymes that when compared to dimeric or monomeric enzymes, hexamerization ensured structural integrity as a prerequisite for thermal stability and activity

Hexamerization and thermostability emerged very early during geranylgeranylglyceryl phosphate synthase evolution

A large number of archaea live in hyperthermophilic environments. In consequence, their proteins need to adopt to these harsh conditions, including the enzymes that catalyze the synthesis of their membrane ether lipids. The enzyme that catalyzes the formation of the first ether bond in these lipids, geranylgeranylglyceryl phosphate synthase (GGGPS), exists as a hexamer in many hyperthermophilic archaea, and a recent study suggested that hexamerization serves for a fine‐tuning of the flexibility – stability trade‐off under hyperthermophilic conditions. We have recently reconstructed the sequences of ancestral group II GGGPS enzymes and now present a detailed biochemical characterization of nine of these predecessors, which allowed us to trace back the evolution of hexameric GGGPS and to draw conclusions about the properties of extant GGGPS branches that were not accessible to experiments up to now. Almost all ancestral GGGPS proteins formed hexamers, which demonstrates that hexamerization is even more widespread among the GGGPS family than previously assumed. Furthermore, all experimentally studied ancestral proteins showed high thermostability. Our results indicate that the hexameric oligomerization state and thermostability were present very early during the evolution of group II GGGPS, while the fine tuning of the flexibility – stability trade‐off developed very late, independent of the emergence of hexamerization

Identification of acetylated diether lipids in halophilic Archaea

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea‐type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn‐glycerol 1‐phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O‐acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first‐time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects

Worldwide Disparities in Recovery of Cardiac Testing 1 Year Into COVID-19

FUNDING SUPPORT AND AUTHOR DISCLOSURES Dr Williams is supported by the British Heart Foundation (FS/ICRF/ 20/26002). Dr Einstein has received speaker fees from Ionetix; has received consulting fees from W. L. Gore & Associates; has received authorship fees from Wolters Kluwer Healthcare – UpToDate; and has received grants or grants pending to his institution from Attralus, Canon Medical Systems, Eidos Therapeutics, GE Healthcare, Pfizer, Roche Medical Systems, W. L. Gore & Associates, and XyloCor Ther- apeutics. Dr Williams has received speaker fees from Canon Medical Systems. Dr Dorbala has received honoraria from Pfizer and GE Healthcare; and has received grants to her institution from Pfizer and GE Healthcare. Dr Sinitsyn has received congress speaker honoraria from Bayer, GE Healthcare, Siemens, and Philips. Dr Kudo has received research grants from Nihon Medi-physics and FUJIFILM Toyama Chemical. Dr Bucciarelli-Ducci is CEO (part-time) of the So- ciety for Cardiovascular Magnetic Resonance; and has received speaker fees from Circle Cardiovascular Imaging, Bayer, and Siemens Healthineers. All other authors have reported that they have no re- lationships relevant to the contents of this paper to disclose.Peer reviewedPublisher PD

International Impact of COVID-19 on the Diagnosis of Heart Disease

BACKGROUND The coronavirus disease 2019 (COVID-19) pandemic has adversely affected diagnosis and treatment of noncommunicable diseases. Its effects on delivery of diagnostic care for cardiovascular disease, which remains the leading cause of death worldwide, have not been quantified. OBJECTIVES The study sought to assess COVID-19's impact on global cardiovascular diagnostic procedural volumes and safety practices. METHODS The International Atomic Energy Agency conducted a worldwide survey assessing alterations in cardiovascular procedure volumes and safety practices resulting from COVID-19. Noninvasive and invasive cardiac testing volumes were obtained from participating sites for March and April 2020 and compared with those from March 2019. Availability of personal protective equipment and pandemic-related testing practice changes were ascertained. RESULTS Surveys were submitted from 909 inpatient and outpatient centers performing cardiac diagnostic procedures, in 108 countries. Procedure volumes decreased 42% from March 2019 to March 2020, and 64% from March 2019 to April 2020. Transthoracic echocardiography decreased by 59%, transesophageal echocardiography 76%, and stress tests 78%, which varied between stress modalities. Coronary angiography (invasive or computed tomography) decreased 55% (p < 0.001 for each procedure). In multivariable regression, significantly greater reduction in procedures occurred for centers in countries with lower gross domestic product. Location in a low-income and lower-middle-income country was associated with an additional 22% reduction in cardiac procedures and less availability of personal protective equipment and telehealth. CONCLUSIONS COVID-19 was associated with a significant and abrupt reduction in cardiovascular diagnostic testing across the globe, especially affecting the world's economically challenged. Further study of cardiovascular outcomes and COVID-19-related changes in care delivery is warranted

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Effekter av patientundervisning

Många hälsoproblem har samband med livsstil. Känslor, problem och konflikter aktualiseras under en sjukdomskris. Patientundervisning är en viktig uppgift som sjuksköterskan har ansvar för. Undervisning går ut på att lära människor om sjukdomens diagnos, behandling och konsekvenser för livet. Patienterna informeras om hur sjukdomen kan tänkas progrediera, vad de själva kan göra för att lindra sitt lidande och varifrån de kan få hjälp. Syftet med studien var att beskriva effekter av patientundervisning. Metoden var en systematisk litteraturstudie där litteraturen bildade informationskällan. Litteraturdatan insamlades genom systematiska sökningar i databaserna Cinahl, Pubmed, Medline och Amed samt genom manuell sökning. Insamlingslitteraturen utgjordes av vetenskapliga primärartiklar. Studiens resultat visade att undervisningen kunde leda till följande effekter hos patienterna: minskad smärta, förbättrade fysiologiska mätvärden, sundare levnadsvanor, ökad självständighet, förhöjd livskvalitet, minskad oro och ökad tillfredsställelse med undervisningen. Resultatet visade att patientundervisningen kunde ha ett samband med dessa människors hälsotillstånd genom att den på olika sätt i de flesta fall hade positiv effekt för deras fysiska och psykiska hälsa

Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes inBacillus subtilis

In Archaea, ether lipids play an essential role as the main building blocks of the cellular membrane. Recently, ether lipids have also been discovered in the domain of Bacteria, and the key enzymes that catalyze their synthesis, glycerol-1-phosphate dehydrogenase and heptaprenylglyceryl phosphate synthase, have been described. In Bacillales, heptaprenylglyceryl phosphate does not become linked to a second polyprenyl moiety like ether lipids in Archaea but is dephosphorylated and acetylated. Here, we report on the enzymes that catalyze these reactions. We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. By screening a B. subtilis knock-out library for deficiency in acetylation, the yvoF gene product was identified to be the acetyltransferase. The acetyl-CoA-dependent enzyme YvoF is a close relative of maltose O-acetyltransferase (MAT). Its catalytic properties were analyzed and compared with MAT. YvoF and MAT partially overlap in substrate and product range in vitro, but MAT is not able to complement the yvoF knock-out in vivo

Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures

The cell membranes of all archaea contain ether lipids, and a number of archaea are hyperthermophilic. Consequently, the enzymes that catalyze the synthesis of membrane ether lipids had to adopt to these rough conditions. Interestingly, the enzyme that establishes the first ether bond in these lipids, the geranylgeranylglyceryl phosphate synthase (GGGPS), forms hexamers in many hyperthermophilic archaea, while also dimeric variants of this enzyme exist in other species. We used Methanothermobacter thermautotrophicus GGGPS (mtGGGPS) as a model to elucidate the benefit of hexamerization. We studied the oligomerization interfaces in detail by introducing disturbing mutations and subsequently compared the stability and activity of generated dimeric and monomeric variants with the wild-type enzyme. Differential scanning calorimetry revealed a biphasic denaturation of mtGGGPS. The temperature of the first transition varies and rises with increasing oligomerization state. This first phase of denaturation leads to catalytic inactivation, but CD spectroscopy indicated only minor changes on the secondary structure level. The residual part of the fold is extremely thermostable and denatures in a second phase at temperatures >120 degrees C. The analysis of another distant native GGGPS enzyme affirms these observations. Molecular dynamics simulations revealed three structural elements close to the substrate binding sites with elevated flexibility. We assume that hexamerization might stabilize these structures, and kinetic studies support this hypothesis for the binding pocket of the substrate glycerol 1-phosphate. Oligomerization might thus positively affect the thermostability-flexibility trade-off in GGGPS by allowing a higher intrinsic flexibility of the individual protomers

Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase

For evolutionary studies, but also for protein engineering, ancestral sequence reconstruction (ASR) has become an indispensable tool. The first step of every ASR protocol is the preparation of a representative sequence set containing at most a few hundred recent homologs whose composition determines decisively the outcome of a reconstruction. A common approach for sequence selection consists of several rounds of manual recompilation that is driven by embedded phylogenetic analyses of the varied sequence sets. For ASR of a geranylgeranylglyceryl phosphate synthase, we additionally utilized FitSS4ASR, which replaces this time-consuming protocol with an efficient and more rational approach. FitSS4ASR applies orthogonal filters to a set of homologs to eliminate outlier sequences and those bearing only a weak phylogenetic signal. To demonstrate the usefulness of FitSS4ASR, we determined experimentally the oligomerization state of eight predecessors, which is a delicate and taxon-specific property. Corresponding ancestors deduced in a manual approach and by means of FitSS4ASR had the same dimeric or hexameric conformation; this concordance testifies to the efficiency of FitSS4ASR for sequence selection. FitSS4ASR-based results of two other ASR experiments were added to the Supporting Information. Program and documentation are available at https://gitlab.bioinf.ur.de/hek61586/FitSS4ASR