Auswahl der wissenschaftlichen Literatur zum Thema „Phosphatidylinositol-4-phosphate 5-kinase de type 1“

Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three enzymes PI5P4Kα, PI5P4Kβ, and PI5P4Kγ, and these enzymes have been implicated in metabolic control, growth control, and a variety of stress responses. Here, we show that mice with germline deletion of type 2 phosphatidylinositol-5-phosphate 4-kinase gamma (Pip4k2c), the gene encoding PI5P4Kγ, appear normal in regard to growth and viability but have increased inflammation and T-cell activation as they age. Immune cell infiltrates increased in Pip4k2c−/− mouse tissues. Also, there was an increase in proinflammatory cytokines, including IFNγ, interleukin 12, and interleukin 2 in plasma of Pip4k2c−/− mice. Pip4k2c−/− mice had an increase in T-helper-cell populations and a decrease in regulatory T-cell populations with increased proliferation of T cells. Interestingly, mammalian target of rapamycin complex 1 (mTORC1) signaling was hyperactivated in several tissues from Pip4k2c−/− mice and treating Pip4k2c−/− mice with rapamycin reduced the inflammatory phenotype, resulting in a decrease in mTORC1 signaling in tissues and a decrease in proinflammatory cytokines in plasma. These results indicate that PI5P4Kγ plays a role in the regulation of the immune system via mTORC1 signaling.

The kinase complex mechanistic target of rapamycin 1 (mTORC1) plays an important role in controlling growth and metabolism. We report here that the stepwise formation of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) regulates the cell type–specific activation and localization of mTORC1. PI(3)P formation depends on the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, as well as the class III PI3K Vps34, while PI(3,5)P2 requires the phosphatidylinositol-3-phosphate-5-kinase PIKFYVE. In this paper, we show that PIKFYVE and PI3K-C2α are necessary for activation of mTORC1 and its translocation to the plasma membrane in 3T3-L1 adipocytes. Furthermore, the mTORC1 component Raptor directly interacts with PI(3,5)P2. Together these results suggest that PI(3,5)P2 is an essential mTORC1 regulator that defines the localization of the complex.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.

Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [γ-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIβ and α) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIβ phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. ( Journal of Biomolecular Screening 2009:838-844)

Insulin secretion from β-cells is regulated by a complex signaling network. Our earlier study has reported that Rac1 participates in glucose- and cAMP-induced insulin secretion probably via maintaining a functional actin structure for recruitment of insulin granules. Type Iα phosphatidylinositol-4-phosphate 5-kinase (PIP5K-Iα) is a downstream effector of Rac1 and a critical enzyme for synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2). By using an RNA interference technique, PIP5K-Iα in INS-1 β-cells could be specifically knocked down by 70–75%. PIP5K-Iα knockdown disrupted filamentous actin structure and caused changes in cell morphology. In addition, PIP2 content in the plasma membrane was reduced and the glucose effect on PIP2 was abolished but without affecting glucose-induced formation of inositol 1,4,5-trisphosphate. At basal conditions (2.8 mm glucose), PIP5K-Iα knockdown doubled insulin secretion, elevated glucose metabolic rate, depolarized resting membrane potential, and raised cytoplasmic free Ca2+ levels ([Ca2+]i). The total insulin release at high glucose was increased upon PIP5K-Iα knockdown. However, the percent increment of insulin secretion by high glucose and forskolin over the basal release was significantly reduced, an effect more apparent on the late phase of insulin secretion. Metabolism and [Ca2+]i rises at high glucose were also attenuated in cells after PIP5K-Iα knockdown. In contrast, PIP5K-Iα knockdown had no effect on cell growth and viability. Taken together, our data suggest that PIP5K-Iα may play an important role in both the proximal and distal steps of signaling cascade for insulin secretion in β-cells.

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

La formation des particules du VIH-1 résulte de l'assemblage des précurseurs Gag sur le feuillet interne de la membrane plasmique (MP) des cellules infectées. Les protéines Gag sont spécifiquement adressées à la MP grâce à des interactions entre le domaine MA et le PI(4,5)P2. Cette étude décrit le rôle des phosphadidylinosito1-4-phosphate 5-kinase de type 1 (PIP5K1alpha, beta et sigma) au cours des étapes tardives du VIH-1 dans le modèle celllulaire HeLa. Nous avons démontré que la PIP5K1alpha est l'enzyme principalement impliquée dans la production du PI(4,5P2. En suivant le trafic de Gag à la MP. Leur inhibition respective induit l'accumulation des précurseurs viraux dans les compartiments intracellulaires distincts et diminue la libération des pseudo-particules Gag dans les deux cas. L'ensemble de nos résultats démontre pour la première fois l'importance de l'activité des PIP5K1alpha et sigma dans l'assemblage et le bourgeonnement du VIH-1 et fournit de nouveaux éléments utiles à la compréhension des étapes tardives du cycle de multiplication viral
The production pocesses of HIV-1 particle of HIV-1 particles results from the assembly of Gag Precursors at the inner leaflet of the plasma membrane (PM) of infected cells. Gag proteins are specifically targeted to PM through interactions between MA domain and PI(4,5)P2. This study describes the role of phosphatidylinositol-4-phosphate 5-kinase type 1 (PIP5K1alpha, beta et sigma) in the late stages of HIV-1 in the context of HeLa cells. We determined that PIP5K1alpha is the principal producer of cellular PI(4,5)P2. By using a confocal microscopy approach, we followed the Gag proteins trafficking and showed that only alpha and y isoforms are required for the correct targeting of Gag to PM. Their respective inhibition leads to the accumulation of viral precursors at distinct intracellular comprtements, and decreases the release of Gag pseudoparticles in both cases. Altogether, our results highlight for the first time the crucial role PIP5K1alpha and sigma in the HIV-1 assembly and budding and provide new insights for a better understanding of the late stages of the virus replication cycle

Type 2 phosphatidylinositol-5-phosphate 4-kinase (PI5P4K) converts phosphatidylinositol-5-phosphate to phosphatidylinositol-4,5-bisphosphate. Mammals have three genes, PIP4K2A, PIP4K2B and PIP4K2C that encode the enzymes PI5P4Kα, PI5P4Kβ and PI5P4Kγ respectively. Studies in mice showed that PI5P4Kβ is a negative regulator of insulin signaling (Lamia et al., 2004) and that co-deletion of Pip4k2b and Trp53 resulted in synthetic embryonic lethality (Emerling et al., 2013). Also, deletion of two alleles of Pip4k2a and one allele of Pip4k2b suppressed the appearance of tumors in Trp53-/- mice. These studies suggest that drugs targeting PI5P4Kα and β could be effective therapies for treating insulin resistance, type 2 diabetes and TP53 mutant cancers. While less is known about PI5P4Kγ, several genome-wide association studies have revealed a SNP in front of the PIP4K2C at an autoimmunity susceptibility loci (Raychaudhuri et al., 2008). To evaluate the role of PI5P4Kγ, I generated Pip4k2c-/- mice and found an inflammatory phenotype with increased tissue immune infiltrates and pro-inflammatory cytokines, correlating with increased helper T cells and decreased regulatory T cells. Also, Pip4k2c-/- mice exhibited upregulated mammalian target of rapamycin complex 1 (mTORC1) signaling in tissues and rapamycin treatment reduced the inflammation of these mice. These studies support the concept that the SNP identified at the PIP4K2C locus in human patients with autoimmunity contributes to disease by reducing expression of PI5P4Kγ and indicates that inhibition of mTORC1 would be beneficial to these patients. Finally, in collaboration with Dr. Nathanael Gray’s laboratory we identified small molecules that covalently react with PI5P4Ks and thereby cause irreversible inhibition. These compounds, PIP4Kin1 and PIP4Kin2 mimicked the effect of shRNA mediated knockdown or knockout of PI5P4Kα and PI5P4Kβ, and impaired the growth of several TP53 mutant cancer cell lines, with little effect on most TP53 wild type cell lines. Utilizing the xenograft tumor model with BT474 (TP53 mutant) and MCF7 (TP53 wild type) cells, we showed that daily treatment of the mice with PIP4Kin2 inhibited the growth of the BT474 tumors but not the MCF7 tumors, without causing any obvious toxicity. These results further validate PI5P4Kα and PI5P4Kβ as targets for treating TP53 mutant cancers.
Medical Sciences

Type I PIP kinases (phosphatidylinositol 4-phosphate 5-kinases, PIP5Ks) catalyse the majority of cellular synthesis of PI(4,5)P2. To date, three mammalian isoforms (r1, r2, r3) have been found. PIP5KIr is subject to complex C-terminal splice variation, enhancing its transcriptional diversity through evolution and producing at least 5 known spliceoforms in the mammals. This study addresses several important questions. (1) Several remarkable differences have been discovered between the neuronal splice variant PIP5KIr_i3 and its close variant, Ir_i2, whose peptide lacks a 26-AA insert near its C-terminus. This study attempts to map these behavioural differences onto motifs within the peptide insert. Furthermore, a site of point mutation is identified near the activation loop, which amplifies the above differences. (2) This study documents properties of the more recently discovered PIP5KIr_i3, about which relatively little is known, for example, the regulation of its subcellular localisation, kinase activity and post-translational modifications. By site-directed mutagenesis and examining more closely several crucial motifs, insight is gained into the putative relationship between the enzyme’s phosphorylation state, cellular localisation, lipid kinase activity and autophosphorylation. (3) The discovery of a new PIP5KIr splice variant, Ir_v6, is described. First discovered in rodents, PIP5KIr_i6 encompasses the 26-AA insert of Ir_i3, but lacks the common C-terminus of Ir_i2 and Ir_i3 which contains peptide motifs that have several roles in vivo. A polyclonal antibody against the C-terminus of Ir_i6 was also developed. Preliminary characterisation of Ir_i6 demonstrates a similar subcellular localisation, but a wider expression profile than its close relative, Ir_i3, suggesting potentially differential functions across tissues and at various developmental stages. (4) The existence of Ir_v3 and Ir_v6 is also confirmed in humans. In light of recent findings of other novel human spliceoforms, this is shown to be a case of intra-exonic splicing producing “alternative 5’ splice site” exons in the human. Overall, this thesis should help to better understand the regulation and physiological roles of PIP5KIr and, specifically, its different splice variants.

Thesis (M.S.)--University of Wisconsin--Madison, 2005.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 81-86).

Regulated secretion of hormones, digestive enzymes and other biologically active molecules requires formation of secretory granules. However, the molecular machinery required for secretory granule biogenesis is incompletely understood. I used powerful genetic approaches available in the fruit fly Drosophila melanogaster to investigate the factors required for biogenesis of mucin-containing ‘glue granules,’ which form within epithelial cells of the third-instar larval salivary gland. I discovered that clathrin and the clathrin adaptor protein complex (AP-1), as well the enzyme type II phosphatidylinositol 4-kinase (PI4KII), are indispensable for glue granule biogenesis. Clathrin and AP-1 are necessary for maturation of exocrine, endocrine and neuroendocrine secretory granules in mammalian cells. I found that Drosophila clathrin and AP-1 colocalize at the TGN and that clathrin recruitment requires AP-1. I further showed that clathrin and AP-1 colocalize with secretory cargo at the TGN and on glue granules. Finally, I demonstrated that loss of clathrin or AP-1 leads to a profound block in secretory granule biogenesis. These findings establish a novel role for AP-1/clathrin-dependent trafficking in the formation of mucin-containing secretory granules. Type II phosphatidylinositol 4-kinase (PI4KII) generates the membrane lipid phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network and is required to recruit cargo to endosomes in mammalian cells. I generated null mutations in the sole Drosophila PI4KII and demonstrated a role for PI4KII in both glue granule and pigment granule biogenesis. PI4KII mutant salivary gland cells exhibit small glue granules and mislocalize glue protein to abnormally large late endosomes. Additionally, PI4KII mutants exhibit altered distribution of the granule specific SNARE, SNAP-24. These data point to a crucial role for PI4KII in sorting of regulated secretory products during granule biogenesis. Together, my results indicate that the larval salivary gland is a valuable system for investigating molecular mechanisms involved in secretory granule biogenesis, and provide a framework for future studies using this system.

Renal osteodystrophy (ROD) is a term that encompasses the various consequences of chronic kidney disease (CKD) for the bone. It has been divided into several entities based on bone histomorphometry observations. ROD is accompanied by several abnormalities of mineral metabolism: abnormal levels of serum calcium, phosphorus, parathyroid hormone (PTH), vitamin D metabolites, alkaline phosphatases, fibroblast growth factor-23 (FGF-23) and klotho, which all have been identified as cardiovascular risk factors in patients with CKD. ROD can presently be schematically divided into three main types by histology: (1) osteitis fibrosa as the bony expression of secondary hyperparathyroidism (sHP), which is a high bone turnover disease developing early in CKD; (2) adynamic bone disease (ABD), the most frequent type of ROD in dialysis patients, which is at present most often observed in the absence of aluminium intoxication and develops mainly as a result of excessive PTH suppression; and (3) mixed ROD, a combination of osteitis fibrosa and osteomalacia whose prevalence has decreased in the last decade. Laboratory features include increased serum levels of PTH and bone turnover markers such as total and bone alkaline phosphatases, osteocalcin, and several products of type I collagen metabolism products. Serum phosphorus is increased only in CKD stages 4-5. Serum calcium levels are variable. They may be low initially, but hypercalcaemia develops in case of severe sHP. Serum 25-OH-vitamin D (25OHD) levels are generally below 30 ng/mL, indicating vitamin D insufficiency or deficiency. The international KDIGO guideline recommends serum PTH levels to be maintained in the range of approximately 2-9 times the upper normal normal limit of the assay and to intervene only in case of significant changes in PTH levels. It is generally recommended that calcium intake should be up to 2 g per day including intake with food and administration of calcium supplements or calcium-containing phosphate binders. Reduction of serum phosphorus towards the normal range in patients with endstage kidney failure is a major objective. Once sHP has developed, active vitamin D derivatives such as alfacalcidol or calcitriol are indicated in order to halt its progression.

Renal osteodystrophy (ROD) is a term that encompasses the various consequences of chronic kidney disease (CKD) for the bone. It has been divided into several entities based on bone histomorphometry observations. ROD is accompanied by several abnormalities of mineral metabolism: abnormal levels of serum calcium, phosphorus, parathyroid hormone (PTH), vitamin D metabolites, alkaline phosphatases, fibroblast growth factor-23 (FGF-23) and klotho, which all have been identified as cardiovascular risk factors in patients with CKD. ROD can presently be schematically divided into three main types by histology: (1) osteitis fibrosa as the bony expression of secondary hyperparathyroidism (sHP), which is a high bone turnover disease developing early in CKD; (2) adynamic bone disease (ABD), the most frequent type of ROD in dialysis patients, which is at present most often observed in the absence of aluminium intoxication and develops mainly as a result of excessive PTH suppression; and (3) mixed ROD, a combination of osteitis fibrosa and osteomalacia whose prevalence has decreased in the last decade. Laboratory features include increased serum levels of PTH and bone turnover markers such as total and bone alkaline phosphatases, osteocalcin, and several products of type I collagen metabolism products. Serum phosphorus is increased only in CKD stages 4-5. Serum calcium levels are variable. They may be low initially, but hypercalcaemia develops in case of severe sHP. Serum 25-OH-vitamin D (25OHD) levels are generally below 30 ng/mL, indicating vitamin D insufficiency or deficiency. The international KDIGO guideline recommends serum PTH levels to be maintained in the range of approximately 2-9 times the upper normal normal limit of the assay and to intervene only in case of significant changes in PTH levels. It is generally recommended that calcium intake should be up to 2 g per day including intake with food and administration of calcium supplements or calcium-containing phosphate binders. Reduction of serum phosphorus towards the normal range in patients with endstage kidney failure is a major objective. Once sHP has developed, active vitamin D derivatives such as alfacalcidol or calcitriol are indicated in order to halt its progression.

We invoke the term semiorganic to describe a new class of nonlinear optical compounds composed of part organic and part inorganic constituents. The recently reported crystal 1-arginine phosphate (LAP) is one such material where organic 1-arginine molecules form cations and crystallize with inorganic phosphate anions.1 We present another type of semiorganic crystal where organic molecules attach as ligands to the inorganic metal salt. We have grown two semiorganic crystals: zinc tris (thiourea) sulfate [Zn(TU)3SO4]2 and (4-nitropyri- dine-N-oxide) mercury chloride [(NPO)HgCl2]. Good optical quality crystals were grown by slow evaporation from solution with dimensions typically ~5 mm on a side. We have carried out nonlinear optical characterization of these materials using degenerate four-wave mixing experiments with a doubled Q-switched Nd:YAG laser (λ = 532 nm) to determine the magnitude of the third-order nonlinear susceptibility x(3). The preliminary results indicate that cobalt doped Zn(TU)3SO4 has a X(3) of about twice that of CS2. We have also observed a significant four-wave mixing signal in (NPO)HgCl2. Both of these crystals show relatively high damage thresholds of up to 17 J/cm2 with 8-ns pulses. Finally, we note that semiorganic crystals have great potential as nonlinear optical materials compared with organic crystals due to their ease of growth to relatively large dimensions, superior mechanical and thermal properties, high optical quality, and high damage thresholds.

Background and aims: In 2020 a global pandemic was declared caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). The pandemic is still ongoing and continues to cause considerable mortality and morbidity world-wide and new variants of the virus are emerging. Rapid development and rollout of vaccines for SARS-CoV-2 is in progress to counter the pandemic but has been tempered by the emergence of new SARS-CoV-2 variants, many of which exhibit reduced vaccine effectiveness. To date there is no approved antiviral treatment for coronavirus disease 2019 (COVID-19). Several studies have shown that Manuka honey has virucidal/antiviral effect. Methylglyoxal (MG), a bioactive component in Manuka honey, has antiviral activity in vitro. MG may modify arginine residues in the functional domains of viral spike and nucleocapsid proteins, resulting in loss of charge, protein misfolding and inactivation. The aim of this study was to characterize the antiviral activity of Manuka honey against SARS-CoV-2 in vitro Materials and methods: Wild-type SARS-CoV-2 with titers of multiplicities of infection (MOI) 0.1 and 0.05 were incubated with 2-fold serial dilutions of 250+ Manuka honey (equivalent to 250 to 31 µM) in infection medium (Dulbecco's Modified Eagle Medium + 2% fetal bovine serum + 100 units/ml penicillin + 100 µg/ml streptomycin) for 3 h. Manuka honey treated and untreated control SARS-CoV-2 was incubated with confluent cultures of Vero cells in vitro for 1 h, cultures washed with phosphate-buffered saline and incubated in fresh infection medium at 37°C for 4 - 5 days until 70% of virus control cells displayed cytopathic effect. We also studied the effect of scavenging MG in Manuka Honey with aminoguanidine (AG; 500 µM) on virucidal activity. The antiviral activity of MG was judged by median tissue culture infectious dose (TCID50) assays. Data analysis was by logistic regression. TCID50 (mean ± SD) was deduced by interpolation. Results: Diluted Manuka honey inhibited SARS-CoV-2 replication in Vero cells. SARS-CoV-2 was incubated in diluted Manuka honey in medium at 37°C for 3 h before adding to Vero cells. Manuka honey dilutions down to 125 µM MG equivalents completely inhibited cytopathic effect of SARS-CoV-2 whereas 31.25 µM and 62.5 µM MG equivalents had limited effect. Logistic regression and interpolation of the cytopathic effect indicated that the TCID50 = 72 ± 2 µM MG equivalents for MOI of 0.1. Prior scavenging of MG by addition of AG resulted in virus replication levels equivalent to those seen in the virus control without AG. Conclusion: Manuka honey has antiviral activity against SARS-CoV-2 when incubated with the virus in cell-free media at no greater than ca. 40-fold dilutions of 250+ grade. Anti-viral activity was inhibited by AG, consistent with the anti-viral effect being mediated by MG. Manuka honey dilutions in MG equivalents had similar antiviral effect compared to authentic MG, also consistent with MG content of Manuka honey mediating the antiviral effect. Whilst Manuka honey may inactivate SARS-CoV-2 in cell-free culture medium, its antiviral activity in vivo for other than topical application may be limited because of the rapid metabolism of MG by the glyoxalase system and limited bioavailability of oral MG.

In recent years, legislative authorities in the US, Europe and Japan have steadily reduced engine exhaust emissions, i.e., carbon monoxide (CO), hydrocarbons (HC), sulphur, particulate matter (PM) and nitrogen oxides (NOx) to improve air quality. To meet these requirements engine manufacturers have had to make significant design changes and as a consequence new engine lubricant specifications from Industry bodies (ACEA, EMA, JAMA) and individual OEMs have had to be introduced to ensure adequate lubrication of these new engines. This has led to significant changes to heavy-duty diesel engine oil (HDDEO) oil formulation composition. Engine design modifications to increase fuel combustion efficiency such as increased peak cylinder pressure and increased fuel injection pressures have placed higher stress on piston rings and liners, bearings and valve train components [1], and improved oil consumption has meant longer oil residence time in the piston ring belt area. The practice of retarded fuel injection timing and exhaust gas recirculation (EGR) as measures to reduce NOx levels by reducing peak combustion temperature has had a considerable impact on lubricant performance. Retarded injection leads to higher soot levels which can cause valve train wear and piston ring liner wear and soot-induced thickening, whilst EGR leads to increased corrosive acids and wear in the combustion chamber. Currently in Europe, Euro 3 heavy-duty engines predominantly use retarded fuel injection as the primary NOx emission control strategy although there are cases where EGR is used. In the US, cooled EGR is used by most engine manufacturers to meet US 2002 emissions. HDDEO’s contain a combination of performance additives such as overbased metal detergents, dispersants, antiwear agents and antioxidants designed to provide wear protection, engine cleanliness, and control of soot contaminants and oxidation. Other additive components include selected viscosity index (VI) improvers and pour point depressants to provide necessary viscosity characteristics and shear stability, and also anti-foam agents for oil aeration control. To meet the increased demands from low emission engines, the chemical composition of the performance additives has been modified and levels increased. Current HDDEOs optimized to meet US and European specifications contain typically between 1.3 and 1.9%wt sulphated ash, 0.1–0.14%wt phosphorus and 0.3–1.1.wt sulphur. To meet the next generation emission standards, engines will require the use of exhaust after-treatment devices. In Europe, Euro 4 emission reductions for NOx and PM, scheduled for introduction in 2005, will require the use of either selective catalytic reduction, or the use of EGR in combination with a diesel particulate filter (DPF). To meet the US 2007 requirements, higher levels of EGR than currently used, in combination with DPFs, is envisaged by most engine builders. Exhaust after-treatment devices are already used extensively in some applications such as DPFs on city buses in Europe and the US. Further NOx restrictions are scheduled for Euro 5 in 2008 and USA in 2010. NOx absorber systems, although used in gasoline engines, are still under development for heavy-duty diesel engines and may be available for 2010. Some lubricant base oil and additive components from oil consumed in the combustion chamber are believed to adversely affect the performance of after-treatment devices. Ash material from metal detergents and zinc dithiophosphates (ZDTP) can build up in the channels within particulate filters causing blockage and potentially loss of engine power, leading to a need for frequent cleaning maintenance. The role of sulphur and phosphorus in additive components is less clear. Sulphur from fuel can either oxidize to sulphur dioxide and react through to sulphuric acid, which manifests itself as particulate, or can have a poisoning effect on the catalyst itself. However, the role of sulphur containing additives is yet to be established. Phosphorus from ZDTP antiwear components can lead to a phosphate layer being deposited on catalyst surfaces, which may impair efficiency. Concerns from OEMs regarding the possible effects of ash, sulphur and phosphorus has led to chemical limits being introduced in some new and upcoming engine oil specifications. The ACEA E6 sequence restricts sulphated ash to 1.0%wt max, phosphorus to 0.08%wt max and sulphur to 0.3%wt max, while the PC-10 category scheduled for 2007 will have maximum limits of 1.0%wt sulphated ash, 0.12%wt phosphorus and 0.4%wt sulphur. The resulting constraints on the use of conventional overbased metal detergent cleanliness additives and zinc dithiophosphate antiwear additives will necessitate alternative engine oil formulation technologies to be developed in order to maintain current performance levels. Indeed, performance requirements of engine oils are expected to become more demanding for the next generation engines where emissions are further restricted. If absorbers become a major route for NOx reduction, limits on sulphur and phosphorus are likely to be more restrictive. Oil formulations meeting ACEA E6 and PC-10 chemical limits have been assessed in several key critical lubricant specification tests, looking at valve train and piston ring/cylinder liner wear, corrosive wear in bearings, piston cleanliness and soot-induced viscosity control. It is demonstrated that it is possible to achieve MB 228.5 extended oil drain performance and API CI-4 wear, corrosion and piston cleanliness requirements for current US engines equipped with EGR [2], at a sulphated ash level of 1.0%wt, and phosphorus and sulphur levels, (0.05 and 0.17%wt, respectively), considerably lower than these chemical limits. This is achievable by the use of selected low sulphur detergents, optimized primary and secondary antioxidant systems and non-phosphorus containing, ashless supplementary antiwear additives blended in synthetic basestocks. Field trials in several city bus fleets have been conducted to assess engine oil performance and durability using one of these low sulphated ash, phosphorus and sulphur (SAPS) oil formulations and to examine lubricant effects on particulate filter performance. Engine oil durability testing was conducted in bus fleets in Germany and Switzerland. These trials, involving over 100 vehicles, cover a range of engine types, e.g., Daimler Chrysler and MAN Euro 1, 2 and 3 and different fuel types (low sulphur diesel, biodiesel, and compressed natural gas) in some MAN engines. The fleets are fitted with continuously regenerating particulate filters either from new or retrofitted. Oils were tested at standard and extended drain intervals (up to 60 000km). Used oil analysis for iron, copper, lead and aluminium with the low SAPS oil in these vehicles have shown low wear rates in all engine types and comparable with a higher 1.8% ash ACEA E4, E5 quality oil. Soot levels can vary considerably, but oil viscosity is maintained within viscosity grade, even at 8% soot loading. TBN depletion and TAN accumulation rates are low showing significant residual basicity reserve and control of acidic combustion and oxidation products. Buses in Stuttgart and Berlin have been used to investigate lubricant ash effects of engine oil on particulate filter durability. Exhaust back-pressure is routinely measured and DPF filters removed and cleaned when back pressure exceeds 100 mbar. Comparison of rate of back pressure build up as a function of vehicle distance shows reduced back pressure gradients for the low SAPS oil relative to the 1.8%wt ash oil in both engine types looked at. An average reduction in back pressure gradient of 40% was found in buses equipped with OM 906LA engines in Berlin and 25% with OM 457hLA engines at both locations. Examination of the ash content in DPFs has shown a 40% reduction in the quantity of ash with the low SAPS oil. This investigation shows that it is possible to meet current long oil drain requirements whilst meeting chemical limits for future lubricants and provide benefits in DPF durability.