Phospholipase D-Catalyzed Transphosphatidylation for the Synthesis of Rare Complex Phospholipid Species-Hemi-bis(monoacylglycero)phosphate and Bis(diacylglycero)phosphate

Progress in developing synthetic pathways for novel and complex phospholipid species, such as Hemi-bis(monoacylglycero)phosphates (Hemi-BMPs) and bis(diacylglycero)phosphates (BDPs), is essential for expanding the knowledge and availability of rare and uncommon phospholipid species. These structurally complex phospholipid species have recently gained more attention with promising applications, as active pharmaceutical ingredient carriers in multiple COVID-19 vaccines, or biomarkers for numerous lysosomal storage disorders and certain types of cancers. The presented work facilitates the production of a range of structurally diverse Hemi-BMP and BDP products intending to increase the availability and thereby the understanding of the underlying chemistry for these high-valuable compounds. The transphosphatidylation of phosphatidylcholine with a variety of structurally diverse monoacylglycerols and diacylglycerols is proceeded by phospholipase D (PLD) catalysis in a biphasic system. Optimization in regard to enzyme loading (5 U), substrate mole ratio (1:5 mol/mol), temperature (30 degrees C), and aqueous concentration of (18% v/v) afforded the highest conversion for the model transphosphatidylation of phosphatidylcholine with monoolein, yielding 87% in 2 h. The study additionally proposes a reaction mechanism based on molecular simulation, elegantly elaborating the structural constraints (substrate configuration and character of the fatty acid residues) for access to the active site of PLD accordingly for lower yield of BDPs. The successful system designed for the production of high-valuable Hemi-BMP and BDP-analogues demonstrated in this work promises to enhance the understanding of these complex phospholipids, leading to new scientific breakthroughs.

Novel heterozygous PLCG2 variant presenting with very early-onset perianal abscess

Case report Background: Mutations in the PLCG2 gene can cause PLCG2-associated antibody deficiency and immune dysregulation (PLAID) or auto-inflammation with PLCG2-associated antibody deficiency and immune dysregulation (APLAID). PLAID is characterized by urticarial eruptions triggered by evaporative cooling along with cutaneous granulomas. APLAID may present with early-onset skin inflammation and non-infectious granulomas, uveitis, and colitis. Method(s): Case report and literature review. We performed in silico analysis for variants of uncertain significance (VUS). Result(s): A 29-day-old boy presented to emergency department for failure to thrive. He was found to be SARS-CoV2 positive, had an E. coli UTI in the setting of bilateral perinephric masses which subsequently resolved. He also had a perianal soft tissue abscess measuring 4cm in diameter. Mom reported a similar infection when she was age 2. She also reported intermittent diffuse urticaria triggered following perspiration evaporation.Abscess wall histology showed diffuse neutrophil and lymphocytic infiltration, with cultures growing polymicrobial enteric flora. His serum immunoglobulins G, A, M, and E were within reference range. Naive and memory CD4, CD8, CD19 lymphocyte subsets (including NK cells) were also within age-appropriate reference range. He had a normal neutrophil oxidative burst measured using dihydrorhodamine (DHR) flow cytometry following PMA stimulation, which ruled out a diagnosis of chronic granulomatous disease. On evaporative cooling, the patient had a 2mm wheal with surrounding erythema which resolved rapidly with warming. A targeted primary immunodeficiency panel showed a heterozygous VUS in PLCG2, c.688C > G (p.Leu230Val). The variant was absent from major databases and had a calculated CADD score of 17.77. He had symptomatic resolution after completing 3 weeks of ceftriaxone and metronidazole antimicrobials. Given the concern for PLCG2-associated very early-onset inflammatory bowel disease (VEO-IBD), a fecal calprotectin was obtained at 3 months and found to be elevated (157 mcg/g [ < = 49 mcg/g]). However, he had no symptomatic or macroscopic evidence for VEO-IBD. Conclusion(s): Presence of very early onset abscesses has not been previously described in patients with heterozygous PLCG2 deficiency. This case adds to the expanding variable phenotype of PLCG-2-associated immune dysregulation.

COVID-19, Blood Lipid Changes, and Thrombosis.

Although there is increasing evidence that oxidative stress and inflammation induced by COVID-19 may contribute to increased risk and severity of thromboses, the underlying mechanism(s) remain to be understood. The purpose of this review is to highlight the role of blood lipids in association with thrombosis events observed in COVID-19 patients. Among different types of phospholipases A2 that target cell membrane phospholipids, there is increasing focus on the inflammatory secretory phospholipase A2 IIA (sPLA2-IIA), which is associated with the severity of COVID-19. Analysis indicates increased sPLA2-IIA levels together with eicosanoids in the sera of COVID patients. sPLA2 could metabolise phospholipids in platelets, erythrocytes, and endothelial cells to produce arachidonic acid (ARA) and lysophospholipids. Arachidonic acid in platelets is metabolised to prostaglandin H2 and thromboxane A2, known for their pro-coagulation and vasoconstrictive properties. Lysophospholipids, such as lysophosphatidylcholine, could be metabolised by autotaxin (ATX) and further converted to lysophosphatidic acid (LPA). Increased ATX has been found in the serum of patients with COVID-19, and LPA has recently been found to induce NETosis, a clotting mechanism triggered by the release of extracellular fibres from neutrophils and a key feature of the COVID-19 hypercoagulable state. PLA2 could also catalyse the formation of platelet activating factor (PAF) from membrane ether phospholipids. Many of the above lipid mediators are increased in the blood of patients with COVID-19. Together, findings from analyses of blood lipids in COVID-19 patients suggest an important role for metabolites of sPLA2-IIA in COVID-19-associated coagulopathy (CAC).

Carriers of the p.P522R variant in PLCγ2 have a slightly more responsive immune system.

BACKGROUND: The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. METHODS: We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. RESULTS: In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. CONCLUSION: The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

Tracheotomy In Patients With Covid-19

Relevance. The coronavirus rush, which has appeared since December 2019, has an impact on economic, medical, and social development in all countries of the world. There are still no standard diagnostic and therapeutic plans aimed at limiting this infection. Purpose. To determine the therapeutic role of tracheostomy with patients with coronavirus infection. Material. The prospective study of 100 patients with coronavirus infection was carried out on the basis of State Budgetary Healthcare Institution "Interdistrict Multidisciplinary Hospital" in Nartkala city. When patients were admitted to the clinic, they were randomized into 2 groups depending on the therapy: the first group (comparison, n=50) - patients received traditional therapy in intensive care;the second (main, n=50) group - patients underwent tracheostomy in addition to standard therapy. The average age was 56.2+/-4.8 years. The women's average age was 55 years (55.0%), and the men's - 45 years (45.0%). Methods. Determination of the syndrome of endogenous intoxication, taxation of lipid peroxidation intensification. The local microcirculation was investigated by the apparatus LAKK-02. The activity of the coagulation-lytic blood system was found by thrombelastograph TEG 5000. Results. Early COVID-19 infection demonstrates signs of intoxication, oxidative depression, phospholipase activity, microcirculatory and hemostatic disorders. These changes were the cause of the development of life-threatening complications (neurological, pulmonary, cardiac, etc.). They were associated with the type of treatment. Traditional treatment and the use of mask oxygen turned out to be ineffective, since the homeostasis system disorders remained throughout the investigation period. The inclusion of tracheostomy in traditional therapy makes it possible to quickly improve the course of pathology as it purposefully affects the pathogenetic links of the homeostasis system. Conclusion. The use of tracheostomy in the scheme of standard treatment of coronavirus infection allows improving the effectiveness of general therapy of COVID-19 patients, especially with severe forms.Copyright © 2023, Codon Publications. All rights reserved.

METABOLIC BIOLOGICAL MARKERS FOR DIAGNOSING AND MONITORING THE COURSE OF TUBERCULOSIS.

The international biomedical community has been currently facing a need to find a simple and most accessible type of analysis that helps to diagnose tuberculosis (TB) with the maximum reliability even before the onset of clinical manifestations. Tuberculosis results in more deaths than any other pathogen, second only to pneumonia caused by the SARS-CoV-2 virus, but the majority of infected people remain asymptomatic. In addition, it is important to develop methods to distinguish various forms of tuberculosis infection course at early stages and to reliably stratify patients into appropriate groups (persons with a rapidly progressing infection, chronic course, latent infection carriers). Immunometabolism investigates a relationship between bioenergetic pathways and specific functions of immune cells that has recently become increasingly important in scientific research. The host anti-mycobacteria immune response in tuberculosis is regulated by a number of metabolic networks that can interact both cooperatively and antagonistically, influencing an outcome of the disease. The balance between inflammatory and immune reactions limits the spread of mycobacteria in vivo and protects from developing tuberculosis. Cytokines are essential for host defense, but if uncontrolled, some mediators may contribute to developing disease and pathology. Differences in plasma levels of metabolites between individuals with advanced infection, LTBI and healthy individuals can be detected long before the onset of the major related clinical signs. Changes in amino acid and cortisol level may be detected as early as 12 months before the onset of the disease and become more prominent at verifying clinical diagnosis. Assessing serum level of certain amino acids and their ratios may be used as additional diagnostic markers of active pulmonary TB. Metabolites, including serum fatty acids, amino acids and lipids may contribute to detecting active TB. Metabolic profiles indicate about increased indolamine 2.3-di-oxygenase 1 (IDO1) activity, decreased phospholipase activity, increased adenosine metabolite level, and fibrous lesions in active vs. latent infection. TB treatment can be adjusted based on individual patient metabolism and biomarker profiles. Thus, exploring immunometabolism in tuberculosis is necessary for development of new therapeutic strategies.Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.

Phospholipase D-Catalyzed Transphosphatidylation for the Synthesis of Rare Complex Phospholipid Species─Hemi-bis(monoacylglycero)phosphate and Bis(diacylglycero)phosphate

Progress in developing synthetic pathways for novel and complex phospholipid species, such as Hemi-bis(monoacylglycero)phosphates (Hemi-BMPs) and bis(diacylglycero)phosphates (BDPs), is essential for expanding the knowledge and availability of rare and uncommon phospholipid species. These structurally complex phospholipid species have recently gained more attention with promising applications, as active pharmaceutical ingredient carriers in multiple COVID-19 vaccines, or biomarkers for numerous lysosomal storage disorders and certain types of cancers. The presented work facilitates the production of a range of structurally diverse Hemi-BMP and BDP products intending to increase the availability and thereby the understanding of the underlying chemistry for these high-valuable compounds. The transphosphatidylation of phosphatidylcholine with a variety of structurally diverse monoacylglycerols and diacylglycerols is proceeded by phospholipase D (PLD) catalysis in a biphasic system. Optimization in regard to enzyme loading (5 U), substrate mole ratio (1:5 mol/mol), temperature (30 °C), and aqueous concentration of (18% v/v) afforded the highest conversion for the model transphosphatidylation of phosphatidylcholine with monoolein, yielding 87% in 2 h. The study additionally proposes a reaction mechanism based on molecular simulation, elegantly elaborating the structural constraints (substrate configuration and character of the fatty acid residues) for access to the active site of PLD accordingly for lower yield of BDPs. The successful system designed for the production of high-valuable Hemi-BMP and BDP-analogues demonstrated in this work promises to enhance the understanding of these complex phospholipids, leading to new scientific breakthroughs. © 2023 American Chemical Society.

Administration of Exogenous Surfactant and Cytosolic Phospholipase A2alpha Inhibitors may Help COVID-19 Infected Patients with Chronic Diseases

Background: Coronavirus-19 (COVID-19) pandemic is a worldwide public health problem causing 347,070 deaths from December 25, 2019, till May 25, 2020. Phospholipids are structural components of mammalian cytoskeleton and cell membranes. Phosphatidylglycerol is an anionic lipid found in mammalian membranes in low amounts (1-2%) of the total phospholipids. Also, phosphatidylglycerol suppresses viral attachment to the plasma membrane and subsequent replication in lung cells. Phosphatidylglycerol depletion caused by over expression of cytosolic phos-pholipase A2alpha induces lipid accumulation in lung alveoli and promotes acute respiratory distress syndrome (ARDS). An exogenous-surfactant replacement has been successfully achieved in ARDS and improved oxygenation and lung mechanics. Inhibition of cytosolic phospholipase A2alpha impairs an early step of COVID-19 replication. Aim(s): The present study was carried out to explain the correlation between the administration of exogenous artificial surfactant as well as cytosolic phospholipase A2alpha inhibitors to improve oxygenation and lung mechanics and inhibit COVID-19 replication. Method(s): Database research was carried out on Medline, Embase, Cochrane Library, country-spe-cific journals, and following-up WHO reports published between December 25, 2019-May 25, 2020. Result(s): Till 25 May 2020, coronavirus cases were 5,307,298, with 347,070 deathsand 2,314,849 recovered cases. According to the WHO reports, most COVID-19 deaths seen are in people who suffered from other chronic diseases characterized by phospholipidosis and phosphatidylglycerol deficiency, including hypertension, liver, heart, and lung diseases and diabetes. Phospholipases A2 (PLA2) catalyze the cleavage of fatty acids esterified at the sn-2 position of glycerophospholipids leading to enhanced inflammation and lung damage. Also, cytosolic phospholipase A2alpha inhibitors may reduce the accumulation of viral proteins and RNA. In addition, administration of exogenous phospholipid surfactant may help COVID-19 infected patients with ARDS to remove inflammatory mediators. Conclusion(s): The present study showed a relation between phosphatidylglycerol deficiency in COVID-19 infected patients with ARDS and/or chronic diseases and their mortality. These findings also showed an important approach for the prevention and treatment of COVID-19 infections by using cytosolic phospholipase A2alpha inhibitors and exogenous administration of a specific phos-pholipid surfactant.Copyright © 2021 Bentham Science Publishers.

Phosphatidylglycerol-specific phospholipase C from Amycolatopsis sp. NT115 strain: purification, characterization, and gene cloning.

Recently, phosphatidylglycerol (PG) focused on its important role in chloroplast photosynthesis, mitochondrial function of the sperm, an inhibitory effect on SARS-CoV-2 ability to infect naïve cells, and reducing lung inflammation caused by coronavirus disease 2019. To develop an enzymatic PG determination method as the high-throughput analysis of PG, a PG-specific phospholipase C (PG-PLC) was found in the culture supernatant of Amycolatopsis sp. NT115. PG-PLC (54 kDa by SDS-PAGE) achieved the maximal activity at pH 6.0 and 55 °C and was inhibited by detergents, such as Briji35, Tween 80, and sodium cholate, but not by EDTA and metal ions, except for Zn2+. The open reading frame of the PG-PLC gene consisted of 1620 bp encoding 515-amino-acid residues containing the preceding 25-amino-acid residues (Tat signal peptide sequence). The putative amino acid sequence of PG-PLC was highly similar to those of metallophosphoesterases; however, its substrate specificity was completely different from those of known PLCs.

Secretory Phospholipase A2 and Interleukin-6 Levels as Predictive Markers of the Severity and Outcome of Patients with COVID-19 Infections.

Coronavirus disease (COVID-19) has become a global pandemic. COVID-19 patients need immediate diagnosis and rehabilitation, which makes it urgent to identify new protein markers for a prognosis of the severity and outcome of the disease. The aim of this study was to analyze the levels of interleukin-6 (IL-6) and secretory phospholipase (sPLA2) in the blood of patients regarding the severity and outcome of COVID-19 infection. The study included clinical and biochemical data obtained from 158 patients with COVID-19 treated at St. Petersburg City Hospital No. 40. A detailed clinical blood test was performed on all patients, as well as an assessment of IL-6, sPLA2, aspartate aminotransferase (AST), total protein, albumin, lactate dehydrogenase (LDH), APTT, fibrinogen, procalcitonin, D-dimer, C-reactive protein (CRB), ferritin, and glomerular filtration rate (GFR) levels. It was found that the levels of PLA2, IL-6, APTV, AST, CRP, LDH, IL-6, D-dimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild to severe COVID-19 infections. The levels of IL-6 were positively correlated with APTT; the levels of AST, LDH, CRP, D-dimer, and ferritin; and the number of neutrophils. The increase in the level of sPLA2 was positively correlated with the levels of CRP, LDH, D-dimer, and ferritin, the number of neutrophils, and APTT, and negatively correlated with the levels of GFR and lymphocytes. High levels of IL-6 and PLA2 significantly increase the risk of a severe course by 13.7 and 2.24 times, and increase the risk of death from COVID-19 infection by 14.82 and 5.32 times, respectively. We have shown that the blood levels of sPLA2 and IL-6 increase in cases which eventually result in death and when patients are transferred to the ICU (as the severity of COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.

Similarities and differences of chemical compositions and physical and functional properties of adjuvant system 01 and army liposome formulation with QS21.

A vaccine adjuvant known as Adjuvant System 01 (AS01) consists of liposomes containing a mixture of natural congeners of monophosphoryl lipid A (MPL®) obtained from bacterial lipopolysaccharide, and a tree saponin known as QS21. Two vaccines containing AS01 as the adjuvant have been licensed, including a malaria vaccine (Mosquirix®) approved by World Health. Organization and European Medicines Agency for use in sub-Saharan Africa, and a shingles vaccine (Shingrix®) approved by the U.S. Food and Drug Administration. The success of the AS01 vaccine adjuvant has led to the development of another liposomal vaccine adjuvant, referred to as Army Liposome Formulation with QS21 (ALFQ). Like AS01, ALFQ consists of liposomes containing monophosphoryl lipid A (as a synthetic molecule known as 3D-PHAD®) and QS21 as adjuvant constituents, and the polar headgroups of the liposomes of AS01 and ALFQ are similar. We compare here AS01 with ALFQ with respect to their similar and different liposomal chemical structures and physical characteristics with a goal of projecting some of the likely mechanisms of safety, side effects, and mechanisms of adjuvanticity. We hypothesize that some of the side effects exhibited in humans after injection of liposome-based vaccines might be caused by free fatty acid and lysophospholipid released by enzymatic attack of liposomal phospholipid by phospholipase A2 at the injection site or systemically after injection.

Phenotypic changes in low-density lipoprotein particles as markers of adverse clinical outcomes in COVID-19.

BACKGROUND AND AIMS: Low-density lipoprotein (LDL) plasma concentration decline is a biomarker for acute inflammatory diseases, including coronavirus disease-2019 (COVID-19). Phenotypic changes in LDL during COVID-19 may be equally related to adverse clinical outcomes. METHODS: Individuals hospitalized due to COVID-19 (n = 40) were enrolled. Blood samples were collected on days 0, 2, 4, 6, and 30 (D0, D2, D4, D6, and D30). Oxidized LDL (ox-LDL), and lipoprotein-associated phospholipase A2 (Lp-PLA2) activity were measured. In a consecutive series of cases (n = 13), LDL was isolated by gradient ultracentrifugation from D0 and D6 and was quantified by lipidomic analysis. Association between clinical outcomes and LDL phenotypic changes was investigated. RESULTS: In the first 30 days, 42.5% of participants died due to Covid-19. The serum ox-LDL increased from D0 to D6 (p < 0.005) and decreased at D30. Moreover, individuals who had an ox-LDL increase from D0 to D6 to over the 90th percentile died. The plasma Lp-PLA2 activity also increased progressively from D0 to D30 (p < 0.005), and the change from D0 to D6 in Lp-PLA2 and ox-LDL were positively correlated (r = 0.65, p < 0.0001). An exploratory untargeted lipidomic analysis uncovered 308 individual lipids in isolated LDL particles. Paired-test analysis from D0 and D6 revealed higher concentrations of 32 lipid species during disease progression, mainly represented by lysophosphatidyl choline and phosphatidylinositol. In addition, 69 lipid species were exclusively modulated in the LDL particles from non-survivors as compared to survivors. CONCLUSIONS: Phenotypic changes in LDL particles are associated with disease progression and adverse clinical outcomes in COVID-19 patients and could serve as a potential prognostic biomarker.

METABOLIC BIOLOGICAL MARKERS FOR DIAGNOSING AND MONITORING THE COURSE OF TUBERCULOSIS.

The international biomedical community has been currently facing a need to find a simple and most accessible type of analysis that helps to diagnose tuberculosis (TB) with the maximum reliability even before the onset of clinical manifestations. Tuberculosis results in more deaths than any other pathogen, second only to pneumonia caused by the SARS-CoV-2 virus, but the majority of infected people remain asymptomatic. In addition, it is important to develop methods to distinguish various forms of tuberculosis infection course at early stages and to reliably stratify patients into appropriate groups (persons with a rapidly progressing infection, chronic course, latent infection carriers). Immunometabolism investigates a relationship between bioenergetic pathways and specific functions of immune cells that has recently become increasingly important in scientific research. The host anti-mycobacteria immune response in tuberculosis is regulated by a number of metabolic networks that can interact both cooperatively and antagonistically, influencing an outcome of the disease. The balance between inflammatory and immune reactions limits the spread of mycobacteria in vivo and protects from developing tuberculosis. Cytokines are essential for host defense, but if uncontrolled, some mediators may contribute to developing disease and pathology. Differences in plasma levels of metabolites between individuals with advanced infection, LTBI and healthy individuals can be detected long before the onset of the major related clinical signs. Changes in amino acid and cortisol level may be detected as early as 12 months before the onset of the disease and become more prominent at verifying clinical diagnosis. Assessing serum level of certain amino acids and their ratios may be used as additional diagnostic markers of active pulmonary TB. Metabolites, including serum fatty acids, amino acids and lipids may contribute to detecting active TB. Metabolic profiles indicate about increased indolamine 2.3-di-oxygenase 1 (IDO1) activity, decreased phospholipase activity, increased adenosine metabolite level, and fibrous lesions in active vs. latent infection. TB treatment can be adjusted based on individual patient metabolism and biomarker profiles. Thus, exploring immunometabolism in tuberculosis is necessary for development of new therapeutic strategies. Copyright © 2022 Saint Petersburg Pasteur Institute. All rights reserved.

Cryo-EM reveals binding of linoleic acid to SARS-CoV-2 spike glycoprotein, suggesting an antiviral treatment strategy.

The COVID-19 pandemic and concomitant lockdowns presented a global health challenge and triggered unprecedented research efforts to elucidate the molecular mechanisms and pathogenicity of SARS-CoV-2. The spike glycoprotein decorating the surface of SARS-CoV-2 virions is a prime target for vaccine development, antibody therapy and serology as it binds the host cell receptor and is central for viral cell entry. The electron cryo-microscopy structure of the spike protein revealed a hydrophobic pocket in the receptor-binding domain that is occupied by an essential fatty acid, linoleic acid (LA). The LA-bound spike protein adopts a non-infectious locked conformation which is more stable than the infectious form and shields important immunogenic epitopes. Here, the impact of LA binding on viral infectivity and replication, and the evolutionary conservation of the pocket in other highly pathogenic coronaviruses, including SARS-CoV-2 variants of concern (VOCs), are reviewed. The importance of LA metabolic products, the eicosanoids, in regulating the human immune response and inflammation is highlighted. Lipid and fatty-acid binding to a hydrophobic pocket in proteins on the virion surface appears to be a broader strategy employed by viruses, including picornaviruses and Zika virus. Ligand binding stabilizes their protein structure and assembly, and downregulates infectivity. In the case of rhinoviruses, this has been exploited to develop small-molecule antiviral drugs that bind to the hydrophobic pocket. The results suggest a COVID-19 antiviral treatment based on the LA-binding pocket.

Cysteine-Encapsulated Liposome for Investigating Biomolecular Interactions at Lipid Membranes.

The development of a strategy to investigate interfacial phenomena at lipid membranes is practically useful because most essential biomolecular interactions occur at cell membranes. In this study, a colorimetric method based on cysteine-encapsulated liposomes was examined using gold nanoparticles as a probe to provide a platform to report an enzymatic activity at lipid membranes. The cysteine-encapsulated liposomes were prepared with varying ratios of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol through the hydration of lipid films and extrusions in the presence of cysteine. The size, composition, and stability of resulting liposomes were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nuclear magnetic resonance (NMR) spectroscopy, and UV-vis spectrophotometry. The results showed that the increased cholesterol content improved the stability of liposomes, and the liposomes were formulated with 60 mol % cholesterol for the subsequent experiments. Triton X-100 was tested to disrupt the lipid membranes to release the encapsulated cysteine from the liposomes. Cysteine can induce the aggregation of gold nanoparticles accompanying a color change, and the colorimetric response of gold nanoparticles to the released cysteine was investigated in various media. Except in buffer solutions at around pH 5, the cysteine-encapsulated liposomes showed the color change of gold nanoparticles only after being incubated with Triton X-100. Finally, the cysteine-encapsulated liposomal platform was tested to report the enzymatic activity of phospholipase A2 that hydrolyzes phospholipids in the membrane. The hydrolysis of phospholipids triggered the release of cysteine from the liposomes, and the released cysteine was successfully detected by monitoring the distinct red-to-blue color change of gold nanoparticles. The presence of phospholipase A2 was also confirmed by the appearance of a peak around 690 nm in the UV-vis spectra, which is caused by the cysteine-induced aggregation of gold nanoparticles. The results demonstrated that the cysteine-encapsulated liposome has the potential to be used to investigate biological interactions occurring at lipid membranes.

Hyperinflammation, apoptosis, and organ damage.

The cytokine storm (CS) in hyperinflammation is characterized by high levels of cytokines, extreme activation of innate as well as adaptive immune cells and initiation of apoptosis. High levels of apoptotic cells overwhelm the proper recognition and removal system of these cells. Phosphatidylserine on the apoptotic cell surface, which normally provides a recognition signal for removal, becomes a target for hemostatic proteins and secretory phospholipase A2. The dysregulation of these normal pathways in hemostasis and the inflammasome result in a prothrombotic state, cellular death, and end-organ damage. In this review, we provide the argument that this imbalance in recognition and removal is a common denominator regardless of the inflammatory trigger. The complex reaction of the immune defense system in hyperinflammation leads to self-inflicted damage. This common endpoint may provide additional options to monitor the progression of the inflammatory syndrome, predict severity, and may add to possible treatment strategies.

Relapse of Membranous Nephropathy and Renal Sarcoidosis Following Exposure to SARS-CoV-2 Vaccine (Johnson & Johnson)

The role of infectious agents derived antigens including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been recognized as a trigger for development of autoimmune mediated disorders following natural infection or immunization. However, there is a scarcity of reports of occurrence of autoimmune associated kidney disorders or flare ups following exposure to a SARS-CoV-2 vaccine. A 65-year-old female presented to a nephrology clinic for evaluation of worsening renal dysfunction. The patient is well known to have systemic sarcoidosis under complete remission on low dose prednisone and likely membranous nephropathy (no previous kidney biopsy) with mildly elevated phospholipase A2 receptor (PLA2R) antibodies. Her membranous nephropathy was in partial remission on angiotensin receptor blockage, with urine to protein creatinine ratio (UPCR) of 1.5 g/g . Five months after receiving the single dose SARS-CoV-2 vaccine (Johnson & Johnson®), she started having a flare up of her systemic sarcoidosis with worsening joint, skin and respiratory symptoms. Blood chemistry revealed worsening renal dysfunction with elevated creatinine up to 1.7 mg/dL from her baseline of 1.0 mg/dL. UPCR was also elevated at 3.4 g/g. Urine sediment revealed no red blood cells or casts, only several calcium oxalate dihydrate crystals. A kidney biopsy was performed and showed a combination of membranous nephropathy (PLA2R positive) along with granulomatous interstitial nephritis with well-formed epithelioid granulomas characteristic of sarcoidosis. She was started on high dose prednisone and her renal function improved to 1.2 mg/dL, UPCR decreased to 1.8 g/g and serum PLA2R antibodies became undetectable. She is still being monitored. After many years of renal sarcoidosis and membranous nephropathy remission, the relapse of renal disease after receiving the SARS-CoV-2 vaccine (Johnson & Johnson®) suggests the association between receiving the vaccine and the recurrence of renal sarcoidosis and membranous nephropathy.

Identification of a Dual Inhibitor of Secreted Phospholipase A2 (GIIA sPLA2) and SARS-CoV-2 Main Protease.

The development of novel agents to combat COVID-19 is of high importance. SARS-CoV-2 main protease (Mpro) is a highly attractive target for the development of novel antivirals and a variety of inhibitors have already been developed. Accumulating evidence on the pathobiology of COVID-19 has shown that lipids and lipid metabolizing enzymes are critically involved in the severity of the infection. The purpose of the present study was to identify an inhibitor able to simultaneously inhibit both SARS-CoV-2 Mpro and phospholipase A2 (PLA2), an enzyme which plays a significant role in inflammatory diseases. Evaluating several PLA2 inhibitors, we demonstrate that the previously known potent inhibitor of Group IIA secretory PLA2, GK241, may also weakly inhibit SARS-CoV-2 Mpro. Molecular mechanics docking and molecular dynamics calculations shed light on the interactions between GK241 and SARS-CoV-2 Mpro. 2-Oxoamide GK241 may represent a lead molecular structure for the development of dual PLA2 and SARS-CoV-2 Mpro inhibitors.

Egyptian cobra (Naja haje haje) venom phospholipase A2: a promising antiviral agent with potent virucidal activity against simian rotavirus and bovine coronavirus.

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log10 TCID50 and 2.5 log10 TCID50, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.

SECRETORY PHOSPHOLIPASE A2: A BIOMARKER OF INFLAMMATION IN AUTOIMMUNE, BACTERIAL AND VIRAL DISEASES.

Secretory phospholipases A2 (sPLA2) represent a large superfamily of enzymes with a molecular weight of 14-19 kDa, including 15 groups and more than 30 isoforms belonging to four types: secretory (sPLA2), cytosolic (cPLA2), calcium-independent (iPLA2) and lipoprotein-associated phospholipase A2 (LP-PLA2, PAF-AH). Eleven species of secretory sPLA2s (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA, and XIIB) have been found in mammals, performing versatile functions and participating in the pathogenesis of a wide range of diseases. On the one hand, sPLA2 may promote elimination of damaged, apoptotic cells by hydrolyzing membrane phospholipids, and exerts a strong bactericidal and antiviral properties, including pronounced effects against antibiotic-resistant strains of microorganisms. In this regard, the use of sPLA2 may represent a new strategy for the treatment of bacterial and viral infections. Moreover, due to the action of sPLA2 on its substrates, a number of biologically active molecules (arachidonic, lysophosphatidic acids, lysophospholipids, fatty acids, prostaglandins, leukotrienes, thromboxanes) are formed, which provide strong inflammatory, detergent, coagulating effects and increase vascular permeability. This pro-inflammatory role of sPLA2 may explain its increase levels and activity in cardiovascular, respiratory, autoimmune, metabolic, oncological, bacterial and viral disorders. The review article presents a classification of sPLA2 isoforms, their substrates, regulatory factors, biological significance, and mechanisms of their strong bactericidal, virucidal, and proinflammatory activity in the heart and lung disorders, autoimmune, metabolic, bacterial, and viral diseases. In particular, the mechanisms of the selective action of sPLA2 against Gram-positive and Gram-negative microorganisms are discussed. We consider diagnostic and prognostic significance, correlations between elevated levels and activity of sPLA2 and distinct clinical symptoms, severity and outcome in the patients with coronary heart disease (CAD), acute myocardial infarction (AMI), atherosclerosis, acute inflammatory lung injury (ALI), respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, bronchial asthma, bacterial infections, septicemia and viral (COVID-19) infections. The opportunity of using sPLA2 as a biomarker of the severity and outcome of patients with chronic obstructive pulmonary disease, bacterial infections, sepsis and viral infections, including COVID-19, is also considered. (English) [ FROM AUTHOR] Секреторные фосфолипазы А2 (sPLA2) представляют собой большое суперсемейство ферментов с молекулярной массой 14-19 кДа, включающее 15 групп и более 30 изоформ, принадлежащих к четырем типам: секреторный (sPLA2), цитозольный (cPLA2), кальций-независимый (iPLA2) и липопротеин-ассоциированная фосфолипаза A2 (LP-PLA2, PAF-AH). У млекопитающих обнаружены одиннадцать секреторных sPLA2 (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA и XIIB), выполняющие разносторонние функции и участвующие в патогенезе широкого спектра заболеваний. С одной стороны, sPLA2, гидролизуя фосфолипиды мембран, способствуют элиминации поврежденных, апоптотических клеток и оказывают сильное бактерицидное, вируцидное действие, в том числе против антибиотикорезистентных штаммов микроорганизмов. Ð’ этом плане использование sPLA2 может представлять новую стратегию терапии бактериальных и вирусных инфекций. С другой стороны, в результате действия sPLA2 на ее субстраты образуются биологически активные молекулы (арахидоновая, лизофосфатидная кислоты, лизофосфолипиды, жирные кислоты, простагландины, лейкотриены, тромбоксаны), которые оказывают сильное воспалительное, детергирующее, коагулирующее действие и повышают проницаемость сосудов. Такая провоспалительная роль sPLA2 обуславливает повышение ее уровней и активности при сердечно-сосудистых, дыхательных, аутоиммунных, метаболических, онкологических, бактериальных и вирусных заболеваниях. Ð’ обзоре приводится классификация изоформ sPLA2, рассматриваются их субстраты, регулирующие факторы, биологическое значение и механизмы сильного бактерицидного, вируцидного действия, а также провоспалительной активности при сердечно-сосудистых, дыхательных, аутоиммунных, метаболических, бактериальных и вирусных заболеваниях. Отдельно излагаются механизмы селективного действия sPLA2 в отношении грамположительных и грамотрицательных микроорганизмов. Обсуждаются диагностическая, прогностическая значимость, корреляции повышенных уровней и активности sPLA2 с клиническими симптомами, тяжестью и исходом пациентов с ишемической болезнью сердца (CAD), острыминфарктом миокарда (AMI), атеросклерозом, острым воспалительным повреждением легких (ALI), респираторным дистресс-синдромом (ARDS), хронической обструктивной болезнью легких (COPD), ревматоидным Ð°Ñ€Ñ‚Ñ€Ð¸Ñ Ð¾Ð¼, бронхиальной астмой, бактериальными инфекциями, сепсисом и вирусными (COVID-19) инфекциями. Рассматривается возможность использования sPLA2 в качестве биомаркера тяжести и исхода пациентов с хронической обструктивной болезнью легких, бактериальными инфекциями, сепсисом и вирусными, в том числе COVID-19, инфекциями. 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