This review summarizes the research on phospholipids and their use for drug delivery related to the Phospholipid Research Center Heidelberg (PRC). The focus is on projects that have been approved by the PRC since 2017 and are currently still ongoing or have recently been completed. The different projects cover all facets of phospholipid research, from basic to applied research, including the use of phospholipids in different administration forms such as liposomes, mixed micelles, emulsions, and extrudates, up to industrial application-oriented research. These projects also include all routes of administration, namely parenteral, oral, and topical. With this review we would like to highlight possible future research directions, including a short introduction into the world of phospholipids. Phospholipids are unique and versatile molecules. They are of natural occurrence and the main components in cellular membranes. Arranged as a lipid bilayer, phospholipids play a significant role in the structure and functionality of biological membranes. They are amphiphilic and consist of a hydrophilic headgroup and a lipophilic/hydrophobic tail .sn-1 and sn-2 position of the glycerol backbone are esterified with fatty acids of varying length and degree of saturation. The remaining sn-3 position is esterified with phosphoric acid, which, in turn, is esterified with an alcohol [sn-1, sn-2, and sn-3 of the glycerol introduces chirality. Depending on the structure of the polar headgroup and pH of the surrounding medium, PE and PC are zwitterionic and have a neutral charge at pH 7, whereas PG, PI, and PS are negatively charged at this pH value. As an example, the chemical structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the different types with alternative headgroups are shown in In phospholipids, the sn-glycero-3-phosphocholine (DPPC), occurs [The physiological functions of phospholipids are manifold. For instance, besides their functional role in cell membranes, phospholipids, mainly PC, have digestion/metabolic functions in bile  to solubilize cholesterol and fatty components in food and lipophilic drug substances . Moreove, occurs , lowerin, occurs , and it , occurs  and apop, occurs .Due to their physiological roles, phospholipids possess a very low toxicity profile and can be used for any route of administration . The strSince the foundation of the PRC, 90 research projects related to phospholipids have been supported as of 2020. In this comprehensive review, we classify the projects according to the main application route of phospholipid-based formulations in parenteral (51), oral (20), and topical projects (10). In addition, nine projects related to basic phospholipid research are included. The distribution of the projects according to their main application route/basic research as well as the location of the institutes of the principal investigator is provided in However, before performing research with phospholipids, their quality  and the intended use should be taken into consideration. Depending on the type of application of the phospholipids, there are different requirements to prove their pharmaceutical quality.First, one must be aware of the different meanings and uses of the term “phosphatidylcholine” and “lecithin”. According to the United States Pharmacopeia (USP), lecithin is described as “a complex mixture of acetone-insoluble phosphatides , which consist chiefly of PC, PE, PI, and phosphatic acid (PA), present in conjunction with various amounts of other substances such as triglycerides, fatty acids, and carbohydrates, as separated from the crude vegetable oil source. It contains not less than 50% of acetone-insoluble matter.” Unfortunately, especially in the American literature, lecithin is also used as a synonym of PC, which is the pure compound. Descriptions of the phospholipid component in products as “lecithin” leaves open which lipid is used. We therefore recommend using for natural phospholipids the term “lecithin” only when the product contains less than 80% by weight total phospholipids and the term “phospholipid” when the product contains 80–100% by weight phospholipids ; the specific phospholipid should be mentioned when the product contains more than 90% by weight of that specific phospholipid.sn-1 position, namely lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE). In all cases, PC is the main phospholipid component. To convert these materials into high-quality excipients, meeting pharmacopeial and regulatory requirements for parenteral and other specific formulations, extraction and chromatography procedures are applied  seed, wheat germ, and flax seed; and animal material such as hen egg yolk, milk, or krill. The phospholipid and fatty acid profiles of the various lecithins depend, of course, on the raw material sources. The composition of de-oiled vegetable lecithin from variable sources and egg lecithin, respectively, are provided as example in lied see . cis-double bonds: C14:0, myristic acid (tetradecanoic acid); C16:0, palmitic acid (hexadecenoic acid); C18:0, stearic acid (octadecanoic acid); C18:1, oleic acid (octadecenoic acid); C18:2, linoleic acid (octadecadienoic acid); C18:3, α-linolenic acid ; C20:0, arachidic acid (eicosanoic acid); C20:4, arachidonic acid ; C22:0 behenic acid (docosanoic acid); C22:4, docosatetraenoic acid; C22:5, docosapentaenoic acid (DPA); and C22:6, cervonic acid . The presence of the polyunsaturated fatty acids (PUFAs) C20:4 and C22:6 is typical for hen egg yolk phospholipids.The fatty acid composition of typical batches of vegetable de-oiled lecithins and egg phospholipid of different PC content, respectively, is given in Tm) is below 0 °C. These phospholipids are at ambient temperature in the liquid crystalline state (Lα phase) and form upon hydration flexible structures/mesophases [Tm are preferred [Due to the presence of unsaturated fatty acids in natural phospholipids, the liquid crystalline to gel phase transition temperature  is an enzyme with a combination of both PLA1 and PLA2 activities; that is, it can cleave acyl chains from both the sn-1 and sn-2 positions. Phospholipase C (PLC) can hydrolyze the bond between the glycerol oxygen and the phosphate group, leading to the formation of diacylglycerol and phosphocholine. Unfortunately, this enzyme family is only active for the hydrolysis reaction and not for the reformation process. Therefore, PLC and PLB are not used industrially. Finally, phospholipase D (PLD) is the only potential enzyme for polar group modification. This enzyme cleaves the bond between the phosphate and the choline. Examples of enzyme-modified (“semi-synthetic”) natural phospholipids prepared from natural PC are lyso-PC, soybean PE, soybean PG, egg PG, and their saturated analogs.Nowadays, alternative biochemical synthesis routes via enzyme-catalyzed reactions serve as a viable alternative to organic-chemical synthesis steps. In that respect, the use of enzymes for phospholipid modification has moved quickly in recent years, not only in academic research but also in industry . The fasO-ethylphosphatidylcholine [To study more mechanistic biochemical or biophysical aspects of phospholipids at the molecular level in natural environments or in model membranes, various synthetic approaches to chemically well-defined phospholipids have been developed. These “full-synthetic” phospholipids are then homogeneous with respect to the polar headgroup and fatty acid composition ,20,21. Tlcholine , to named-mannitol. However, this synthetic method is lengthy, requires toxic chemicals and solvents in large excess, and reaction intermediates may be unstable and subject to partial racemization [R) or (S) glycidyl tosylates [sn-glycero-3-phosphocholine, l-α-glyceryl-phosphorylcholine, α-GPC, or choline alfoscerate. GPC can be synthesized in an enantioselective manner using a biotransformation procedure based on the phosphorylation of glycerol by adenosine triphosphate (ATP) catalyzed glycerol kinase [For the preparation of “full-synthetic” phospholipids, several starting compounds are conceivable. The classical approach to synthesize diacyl-PCs is to start from mization ,25. A siosylates . The enal kinase  or, prefl kinase ,28, and l kinase  or 2-metl kinase . For theTo exemplify the production process of natural phospholipid excipients starting from plant oil, the production process of soybean lecithin is given. First, crude soybean lecithin is isolated by degumming of the crude soybean oil as obtained by extraction from soybeans  Figure .The crude soybean lecithin obtained serves as starting material for production at a large scale of soybean lecithin fractions with higher PC content. These fractions are obtained in high yields by extraction methods using the non-toxic solvents acetone and ethanol followed by chromatographic purification procedures and appropriate solvent removal methods. All solvents can be recycled and reused. By selecting appropriate sequential extraction and chromatography methods, several lecithin fractions differing in PC content from 20 to 80% up to pure PC (≥98%) and in ratios of phospholipids to non-polar lipids can be reproducibly achieved. This procedure applies to soybean oil and all vegetable oils used to produce lecithin, and high-purity phospholipids/PC. Furthermore, egg phospholipids are isolated from hen egg yolk with similar extraction and chromatography methods as for soybean lecithin.Natural phospholipids are in general well known to regulatory authorities. Moreover, their track record as excipients with very high tolerability and biocompatibility is outstanding. The World Health Organization (WHO) places no limit on the oral intake of lecithin. Furthermore, no limit for the value of acceptable daily intake (ADI) for lecithin as a food additive is given . The pedThe European Commission declares that lecithin is a food additive (E322) “generally permitted for use in foodstuffs” . FurtherAlthough phospholipids on their own do not have an allergy potential, phospholipids derived from soybean and hen egg yolk must be labelled as potentially allergenic because of the soy and egg origin ,38. It iPhospholipids—natural as well as synthetic—are broadly used in pharmaceutical technology as wetting agents, emulsifiers, and builders or components of different lipid mesophases such as liposomes, micelles, mixed micelles, inverted micelles, cubosomes, etc. . These fThe categories of pharmaceutical formulations, which are of interest to study the occurrence of natural and synthetic phospholipids, respectively, are products used for parenteral administration . These aMore details regarding the use of phospholipids in pharmaceutical formulations, including a variety of examples and drug products, can be found in the literature ,9.In the following sections, we give an overview about the research projects that have been approved since 2017 and are currently still ongoing or have recently been completed. There are 37 research projects, which can be subdivided into parenteral (23 projects), oral (7), and topical (4) administration/use of phospholipid-based formulations, and projects covering basic research (3).®—the first nano-drug approved by the FDA in 1995 [www.phospholipid-research-center.com).The most prominent mode of application of lipid-based formulations is the parenteral route, including i.v., i.m., and s.c. administration. Within this parenteral subgroup, liposomes and liposomal formulations, respectively, of active pharmaceutical ingredients (API) play an important role, which is due to but not restricted to the introduction of the Doxil in 1995 . AlthougOver the last decades, liposomes have played—and they still do—a leading role in the field of nanomedicine . HoweverOne possibility to increase the effectiveness of liposomal drug delivery systems is the use of an external trigger to release the liposomal content at the targeted area. As external triggers, one may use focused ultrasound, light, or X-ray.The group of Robbert Jan Kok  employed two types of stimuli-responsive nanocarriers to bring a plant-derived anti-cancer agent, mistletoe-lectin-I (ML-I) in the respective case, to the target cells. The entrapment of ML-I in liposomes protects the bioactive protein against degradation, while also preventing off-target effects caused by the toxin. Since ML-I cannot pass over the intact lipid membrane, two external stimuli were tested. Heat  induced Another trigger can be light. Tatu Lajunen  combined a light activation technology based on indocyanine green ,59 with To improve the management of treatment-resistant cancers, there is a continued need for new strategies to augment radiotherapy outcomes. The use of ultra-small gold particles (nanoclusters) is a promising way to improve radiation absorption by cancer tissues ,63, yet Hejian Xiong from the University of Texas at Dallas (USA) is following a similar approach. He is using a highly photosensitive nanovesicle (liposome) to achieve repeatable and adjustable on-demand local anesthesia in superficial or deep tissues, which consists of gold nanoparticles attached to mechano-responsive liposomes. Mechano-responsiveness means that liposomes release their cargo upon mechanical stress such as shear stress . A classThe use of ligands, which are presented on the surface of liposomes to bring APIs specifically to the sites of action, is a longstanding endeavor for liposome researchers. Although positive results in animal models have been reported, no positive effects on the efficiency in patients have been published for ligand-targeted liposomes so far ,75. ReceUntil encapsulated APIs are released from the liposomal container, the formulation has the pharmacokinetic properties of the nanoparticle and is therefore subject to the enhanced permeability and retention (EPR) effect. Due to the fenestration of endothelial cells in capillary vessels of malignant tumors, liposomes up to a size of 500 nm can diffuse into the tissue and accumulate there ,78. ThisThe project of Luisa Corvo  focuses on liver reperfusion injury, which is a severe pathology with a very high unmet therapeutic need, as patients in whom a liver transplantation fails do not have any treatment option left . The objThe tumor microenvironment strongly contributes to aggravating tumor growth and causing treatment failure. Tumor-associated macrophages (TAMs), M2-type macrophages, are key cells in the tumor microenvironment, inducing tumor growth, invasion, and metastasis ,84. In tIn nature, biological membranes consist of more than fifty percent of (phospho)lipids. These lipids play a central role in cell–cell interactions and tissue recognition. Despite their important biological role, drug delivery systems rarely take advantage of the lipid profile to improve targeting. In fact, in most cases, proteins or sugar groups conjugated to the surface of lipid-based drug delivery systems are used to modulate cell uptake and biodistribution. In preliminary studies, the group of Avi Schroeder  found that the lipid profile of liposomes is enormously important for determining the biological fate of liposomes in cells of breast cancer. Schroeder will now study how lipids can be used to control tissue distribution ,87,88, sPediatric sarcomas account for about 15% of pediatric cancers. These sarcomas often display a highly aggressive behavior with early tendencies for the development of metastasis. Although current treatment regimens, including surgery and chemotherapy, can achieve good response rates, the relapse rate is generally high with an extremely poor prognosis. The aggressive chemotherapies needed to fight relapsed tumors have significant toxicity, generating late side effects, a major complication in pediatric oncology . Here, lCancer accounts for 26% of all deaths worldwide. While the introduction of checkpoint inhibitors has dramatically changed the outlook of some cancers, only a fraction of all cancer patients benefits from these therapies, mainly due to a failed immune cell activation directed to the tumor . ClassicThe last project in this subgroup is related to drug delivery into the brain. Here, the blood–brain barrier (BBB) is a very selective biological barrier protecting the brain. It is formed by endothelial cells of microvessels being characterized by extremely tight junctions and high expression of export proteins. It is impermeable to macromolecules, including biologicals. However, biologicals are of highest interest for the therapy of Alzheimer’s disease . A promiExtracellular vesicles (EVs) are membrane-contained vesicles released in an evolutionally conserved manner by cells ranging from prokaryotes to higher eukaryotes and plants . The impLiver fibrosis is a reversible wound-healing response to repeated and chronic organ injury, characterized by the aberrant accumulation of the extracellular matrix. Scar formation may finally lead to organ dysfunction, cirrhosis, and hepatocarcinoma. The main profibrogenic cells of injured liver, namely hepatic stellate cells (HSC), following activation by specific stimuli undergo phenotypical changes towards myofibroblasts and contribute to the deposition of collagen . To dateIn another project, ribonucleic acid (RNA)-based therapeutics, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and CRISPR  and CRISPR-associated protein 9 (Cas9) components, play an important role. These RNA-based therapeutics have unprecedented therapeutic potential  and holdA third project is also related the drug loading of exosomes but partially in relation to stem cell therapy. Stem cell therapy is investigated in the management of difficult-to-treat or as of yet incurable diseases, such as cardiac infarction, graft-versus-host disease, and Alzheimer’s disease. With few exceptions, the application of living cells for therapeutic purposes has not yet fully found its way into standard clinical practice, in part due to safety issues and an unclear mode of action. Instead, stem cell-derived exosomes have emerged as key mediators of stem cell biological effects and additionally allow for the elimination of the safety concerns associated with living cells. Furthermore, the inherent therapeutic efficacy of exosomes  can be iBesides the above-mentioned projects using stimuli-responsive liposomes, targeted liposomes, or exosomes, there are four more projects dealing with phospholipid liposomes.Mycobacterium tuberculosis-drugs, formed by different liposomes glued together in “multi-compartment” clusters [In the project of Federico Bordi and Simona Sennato , a new liposome-based multi-drug delivery system is developed, loading simultaneously two anti-clusters . The innclusters , thus reThe production of liposomes with high encapsulation efficiency for high molecular weight APIs, which are sensitive towards heat and aggressive conditions of pH, organic solvents, or surfactants, is an unmet need of increasing interest. Here, the group of Hermann Nirschl  developed a novel process using centrifugation of water-in-oil (w/o) emulsions, based on the use of phospholipids as emulsifiers, avoiding the use of aggressive treatments and allowing asymmetric membrane functionalities. While the previous project addressed the characterization of the applied substances to produce liposomes ,120,121,Another project is focused on non-alcoholic steatohepatitis (NASH). Non-alcoholic fatty liver disease (NAFLD), closely associated with obesity and metabolic syndrome affecting 20–35% population worldwide, is the leading indication for liver transplantation. NASH is the most aggressive form of NAFLD that progresses to end-stage liver failure and liver cancer, and there is no curative therapy available . LipidomLastly, the project of Enrico Mastrobattista  aims to lay the foundation for the use of liposomes to stimulate bone regeneration (osteogenesis) by means of mild immune activation. This is nourished by the knowledge that a short-lived inflammation is pivotal for normal bone healing after damage, and by recent findings that local induction of a mild inflammation can lead to de novo bone formation. Factors such as liposome charge and type of phospholipid will be addressed regarding their intrinsic immuno-stimulatory and osteogenic effects. Furthermore, the functionalization of liposomes with synthetic ligands for pattern-recognition-receptors (PRRs), which could enhance their inflammatory activity, will be investigated. PRR modulation has led to clinical efficacy in anticancer drugs or vaccines. However, the usage for musculoskeletal disease is still in its infancy, and it is unknown which PRR ligands have the greatest osteogenic potential. In addition, it is unclear how  liposomes must be applied at the biomaterial–tissue interface. At the practical level, this project strives to demonstrate the feasibility of liposome-stimulated osteogenesis by fabricating a liposome-based implant coating.www.phospholipid-research-center.com).In addition to the large number of liposome-based projects, there are also several studies that use lipid-based formulations for the intended parenteral application, but focusing on other “lipid structures”, such as lipodisks or extrudates, or by pursuing completely different approaches. An overview about these parenteral projects is given in sn-glycero-3-phosphoethanolamine-N-(amino(polyethylene glycol)2000) (DSPE-PEG2000), respectively, showed ideal physical–chemical characteristics in terms of size and polydispersity index, high miRNA encapsulation efficiency, good stability following incubation in serum, and no hemolytic activity [In this category, the first project is related to cancer therapy. Nanocarriers based on cationic lipids have been largely investigated to deliver DNA or RNA in different forms of cancer. Self-assembling nanoparticles (SANPs) based on cationic lipids have been recently developed for the delivery of bisphosphonates in different form of tumors ,130, amoactivity . MoreovePC-bound ω3 fatty acids (ω3-PC) suppress homeostatic pathways with fundamental roles in tumorigenesis, metastasis, and tumor resistance. By attenuating pro-survival Akt activation, ω3-PC delays cell cycle progression and enhances apoptosis. ω3-PC also increases membrane rigidity, impairs cell migration, interferes with cytoskeleton dynamics, and serves as substrate for the biosynthesis of tumor-suppressive lipid mediators. Supported by the PRC, the potential of ω3-PC in overcoming Akt-dependent tumor resistance has been explored by the group of Andres Koeberle  in an earlier project, and it has been found that the cytotoxic activities of distinct bioinspired agents are potentiated by the combination with ω3-PC ,134,135.co-glycolide) (PLGA). In this respect, Marina Kolbina and Roland Bodmeier  are exploring twin-screw extruded phospholipid-based implants for controlled drug delivery [Controlled parenteral drug delivery presents many advantages compared to conventional parenteral formulations. Along with biodegradable polymers, phospholipids have been widely applied in this field, resulting in several commercially available formulations primarily based on liposomes . Solid idelivery . After tdelivery , this foIn a similar context, the project of Karsten Mäder and Annette Meister  is about to develop and characterize anti-inflammatory phospholipid depot formulations including PS- and PG-enriched extrudates and nanofibers, respectively, for the local therapy in brain, ear, and eye. The project is based on the encouraging results  obtainedAnother project is related to contrast agents. Ultrasound contrast agents are comprised of phospholipid-coated gas microbubbles, 1–10 μm in size, that vibrate  in response to ultrasound. However, the vibration of current microbubbles is unpredictable and, thus, a huge problem for the new theranostic applications of microbubbles, which include ultrasound molecular imaging  and drugThe last project in this subgroup is related to phospholipid nanodisks, also termed lipodisks. Katarina Edwards  is exploring the potential of this novel type of lipid-based nanocarrier for the formulation and delivery of (cancer) therapeutic agents. These disk-shaped nanocarriers are stable, biocompatible, and very versatile structures . Due to When phospholipids, for example in the form of liposomes, are administered orally, one must be aware of degradation processes due the conditions found in the GI tract, i.e., low pH values and the presence of lipases and bile salts. The digestion of (phospho)lipids begins in the oral cavity through exposure to lingual lipases, which are secreted in the buccal cavity by the Ebner’s glands located on the tongue. Digestion continues in the stomach through the effects of both lingual and gastric enzymes. The stomach is also the major site for the emulsification of dietary fat and fat-soluble vitamins, with peristalsis as a major contributing factor. Crude emulsions of lipids enter the duodenum as fine lipid droplets and then mix with bile and pancreatic juice to undergo marked changes in chemical and physical structure. Emulsification continues in the duodenum along with hydrolysis and micellization in preparation for the absorption across the intestinal wall . For furDue to the phospholipids’ amphiphilic nature, they can be technically used in oral dosage forms as emulsifier, wetting agent, solubilizer (mixed micelles), and liposome former. In addition, they can be used as matrix material for solid dispersions when solubilization and fast release of the API are required, whereas saturated phospholipids may be perfectly suitable for slow release matrix tablet formulations. The main types of these formulations can be considered for optimization of the oral bioavailability of poorly water-soluble compounds ,43.www.phospholipid-research-center.com).In the following, we discuss PRC-funded projects covering the oral use of such lipid-based formulations . For resDespite the success of liposomes administered parenterally, the delivery of liposomes via the oral route is impeded by various barriers, namely the chemical instability of phospholipids in the GI tract, the loss of integrity of the liposomes due to mechanical instability of the vesicles itself, and the poor permeability of conventional liposomes due to their relatively large size and the presence of various epithelial barriers in the intestine. A very promising approach to improve the chemical and mechanical stability of liposomes in the GI tract is the partly replacement of the classical monopolar phospholipids by bipolar amphiphiles, so-called bolalipids or bolaamphiphiles ,155. TheThermoacidophiles, where they contribute to the outstanding stability of the Archaea against harsh living conditions, such as low pH values, high salt concentrations, and/or high temperatures. However, the use of the natural occurring archaeal lipids is challenging due to the high costs of the cultivation of the Archaea, and isolation of membrane lipids often results in a mixture of TELs varying in their hydrophobic parts. On the other hand, the total synthesis of these archaeal lipids is also time-consuming, expensive, and in most cases only a small amount of product can be obtained. Here, the group of Simon Drescher  is trying to find an easy-to-synthesize bolalipid that can be used for the stabilization of orally administered liposomes. During this project, two novel classes of artificial bolalipids, the glycerol diether bolalipids [Bolalipids are molecules that consists of a long hydrophobic alkyl chain and two hydrophilic headgroups attached to each end. This special class of lipid molecules can be found in the membranes of certain species of Archaea, for example lalipids ,161 and lalipids ,163, werlalipids ,165, andlalipids . HoweverThaumarchaeota, which are abundant in, for example, wastewater treatment plants and the ocean. They are phylogenetically closely related to hyperthermophilic species and produce unique TELs that have evolved to stabilize the membranes at the ambient temperatures at which these organisms live, which is an interesting feature for the use in liposomes. Hence, the aim of this project is to test if environmental sources for TEL-producing Archaea can be exploited in an economical way.The project of Alexander Treusch  is tackling the stability issue of orally administered liposomes from a different perspective. Instead of synthesizing artificial bolalipids or extracting them from cultured species, he is trying to exploit alternative environmental sources where bolalipid-producing Archaea are abundant. While TELs are present in a range of microorganisms of the domain Archaea, only TELs from two hyperthermophilic species have been tested for oral drug delivery by liposomes so far . The maiBesides stabilized liposomes, one can also use oral mixed micellar formulations for drug delivery purposes. In the past, particularly in very young children, liquid dosage forms were the formulations of choice in oral (pediatric) drug administration. Although they have an excellent safety profile, little attention has been paid to the evaluation of phospholipids in pediatric formulation development. Besides ensuring patient adherence and allowing for the administration of flexible doses, pediatric formulation concepts should ensure that the drug is sufficiently bioavailable, and the pediatric dosage form is made of excipients that are proven to be non-toxic in this vulnerable patient group. These requirements are a challenge when an enabling formulation is required to guarantee a robust solubilization in the GI environment of the target patient group. The combination of natural phospholipids and bile salts in designing mixed micelle formulations represents an interesting formulation approach for developing oral pediatric dosage forms for poorly soluble drugs ,168,169.Plasmodium falciparum malaria. Hence, design and development of more effective and affordable drug delivery systems are necessary, as drug resistance can also be combated through the engineering of novel drug delivery systems. The project of Anthony Attama  is focused on the development of a novel delivery system of an antimalarial extract clinically proven to be effective against P. falciparum malaria, together with long-standing use in traditional settings, and an alternative delivery system of artemether/lumefantrine combination (ACT) [Another project is focused on the development of phospholipid-based depot antimalarial tablets. Malaria affects millions of people yearly in malaria-endemic regions of the world, and cases of treatment failure have been recorded with artemisinin combination therapies (ACTs), for example, artemether and lumefantrine, the first-line drug combination for the treatment of on (ACT) ,171. TheBesides the use of tablets and orally administered liposomes, one can also think of amorphous solid dispersions to deliver drugs. Co-amorphous drug lecithin systems are envisaged as both amorphous solid dispersions and lipid-based drug delivery systems. As such, it is hypothesized that they form an excellent basis for both poorly water-soluble lipophilic compounds  and poorly water-soluble compounds with high melting points (“brick dust” molecules), and thus may be broadly applicable. In this context, the project of Thomas Rades  is envisaged to investigate the potential use of lecithin  as a co-Phospholipids may also play an important role with respect to peptide/protein delivery. The oral delivery of peptides is desired in many therapeutic regimes but is challenged by the physical–chemical properties of the peptides, resulting in low absorption. Low gastric pH and proteases distributed in the entire GI tract can degrade orally administrated peptides ,178,179.The last project in this “oral” context is also related to amorphous solid dispersions. The aim of the project by Paulina Skupin-Mrugalska  is the usage of phospholipids as components of mixed phospholipid and polymer-based amorphous solid dispersions for oral delivery of poorly soluble drugs. Selected formulations will be screened by using derivatives of cellulose or polyvinylpyrrolidone and (hydrogenated) phospholipids. Amorphous solid dispersions of drug substance  in a polymer-phospholipid matrix will initially be prepared by simple film-casting/batch-melting and later by hot-melt extrusion. Fenofibrate is commonly used as a model drug for amorphous solid dispersion studies and has been reported as a suitable candidate for hot-melt extrusion manufacturing ,183. ThePhospholipid-based formulations have gained major interest in the field of topical drug delivery due to their physicochemical properties. As amphiphilic constituents of cellular membranes, phospholipids exhibit both high biocompatibility and great emulsifying power. Thus, plentiful phospholipid-based formulations and drug carriers such as liposomes, SLNs, micelles, and organogels have been developed and investigated by different research groups ,187,188.www.phospholipid-research-center.com).There are four projects covering the topical use of lipid-based formulations , which aPhospholipids, especially of plant origin, are excellent excipients for dermal administration. They can be used to promote drug–skin interactions, to enhance the skin barrier, to act as a moisturizer, and to increase the time of residence of a drug substance at the skin surface. Phospholipids are further applied topically, for example in the lung, to the eye, to the vagina, etc., enabling the administration of poorly water-soluble drugs and extending the drug release, respectively. Furthermore, phospholipid liposomes are suitable for the encapsulation of hydrophilic as well as lipophilic drug substances, and these liposomes can be used in different dosage forms such as gels and liquid sprays. There are slightly different effects of phospholipids in or on the skin; whereas formulations with unsaturated phospholipids tend to penetrate deeper into the skin compared to formulations without phospholipids—thereby helping to deliver the drug to the target site—hydrogenated phospholipids, in turn, increase retention time of drugs on the surface of the skin and help to keep the barrier function intact.A topical vaginal therapy presents the non-invasive and direct delivery of drugs to the site of action at lower doses while escaping adverse side effects caused by systemic drug administration. To achieve efficient, safe, and targeted vaginal therapy of sexually-transmitted bacterial diseases and recurrent bacterial vaginosis, the group of Željka Vanić  aims to develop an efficient, safe, and stable liposome-based delivery system containing azithromycin destined for improved topical vaginal therapy of sexually-transmitted diseases and bacterial vaginosis. Encapsulation into liposomes is expected to increase azithromycin solubility and allow fusion with bacterial cells, resulting in an improved localized effect even at lower doses in comparison with the free drug.The human skin serves as a barrier between the body and the environment. Therefore, it is prone to microbial, thermal, mechanical, and chemical threats that can cause acute or chronic wounds. Triterpenes from the outer bark of birch are known for various pharmacological effects, including enhanced wound healing ,190. PolTo increase the knowledge in the field of cytotoxicity of topical formulations and of phospholipids, the project of Claudia Valenta  evaluates different phospholipid formulations  on the cell viability of viable human keratinocytes and fibroblasts, as determined by cell culture toxicity tests. Moreover, the effect of the formulations on skin penetration and permeation of incorporated model drugs is determined ,196—to fAs mentioned above, natural plant-based phospholipids are interesting excipients for the formulation of macro- and micro-emulsions for dermatological as well as cosmetic uses. Many natural phospholipids are available for topical formulations. Although such phospholipids mixtures are well described in terms of headgroup and fatty acid composition, their physicochemical properties are not described in detail to enable a more rational selection for the effective design of topical formulations. In this context, the project of Reinhard Neubert and Gerald Brezesinski  characterizes plant-based hydrogenated (saturated) phospholipids for dermato-pharmaceutical and cosmetic use ,200. Thewww.phospholipid-research-center.com).Finally, there are three phospholipid-based projects, which do not deal with a special form of application/administration but rather with basic research on phospholipids, although it must certainly be said at this point that the boundaries here are “fluid”. The first project in this category by Thomas Gutsmann and Christian Nehls  focuses on the production of liposomes to mimic bacterial membranes using a novel microfluidic platform ,202,203.The next project is really a theoretical one. Despite the generally recognized relevance of applying theoretical concepts to the design of lipid-based drug carrier systems, there is little ongoing research to develop new and further refine existing concepts. The group of Sylvio May  therefore proposes to develop a variety of theoretical models for the interaction between drug molecules and liposomes. Within this project, the influence of membrane-intercalating drug molecules on the stability of liposomes ,205, theThe last project in this context is about the fate of mixed micellar formulations after i.v. administration. Mixed micellar drug delivery systems (MM-DDS) are used to solubilize poorly soluble drugs. They are applicable for oral and parenteral administration, well tolerated, stable upon storage, and easy to produce. MM-DDS have been marketed for decades, but despite all the advantages, the number of products has remained quite limited. A reflection paper issued by the EMA  names seIn addition to the projects described above, there are some new projects that have been approved quite recently. The project by Jai Prakash  about immunostimulating liposomes targeting the M2 macrophage to eradicate cancer follows on seamlessly from the work he has completed (see Despite the relatively simple chemical structure of phospholipids, this review shows how versatile the applications of phospholipids are and how little we know in some areas—which in turn is the reason for the existence and mission of the Phospholipid Research Center Heidelberg. The PRC has set itself the goal of exploring and harnessing the full potential of phospholipids. Phospholipids are already included in numerous approved drug products, but their potential is far from exhausted. Phospholipids are extremely well tolerated—they are biocompatible, biodegradable, and non-toxic—and their capabilities go far beyond those of conventional emulsifiers and solubilizers, respectively. Moreover, phospholipids have multifunctional technological properties as surfactant excipient, being, for example, emulsifier, solubilizer, liposome former, and wetting agent, and they can be applied in any route of administration. In the latter regard, they are superior compared to synthetic, non-biodegradable excipients, which are not always suitable for every route of administration and which are, per se, non-physiological.With this review, we tried to give an overview of the current and PRC-supported research on phospholipids. As one will quickly realize, phospholipids are not the same as liposomes, and the applications of phospholipids are much more diverse. Especially in the field of oral dosage forms, the liposomal structure may not be a prerequisite for solubilizing lipophilic actives, but the phospholipids in the liposomal dosage form will be, after dissolving of the liposomes in the bile and enzymatic degradation by phospholipases, integrated into mixed micelles with bile salts and in this way enhance the solubility of the lipophilic APIs. Nevertheless, there may also be a future for intact liposomes, using bolalipids, which are not susceptible to enzymatic degradation in the gut. Such liposomes may be useful for enhancing the oral absorption of encapsulated APIs. Another important function of phospholipids is their use as emulsifiers in emulsions, especially o/w emulsions, for any route of administration. They compete in that respect for oral and topical administration with alternative synthetic detergents/emulsifiers or polymers. After parenteral administration, they also compete with solubilizing or emulsifying synthetic detergents, but phospholipids do not possess the properties to cause allergic reactions up to anaphylactic shocks, typical for these synthetic detergents, such as the polysorbates. Therefore, research on parenteral administration with phospholipids is in focus, also when taking the number of projects supported by the PRC into consideration.Due to the advent of lipid-based gene therapy, as demonstrated by the product Onpattro, and the vigorous research ongoing in the area of lipid nanoparticles as adjuvants in COVID-19 vaccine candidates, it is to be expected that the area of phospholipids for parenteral use will be extended with many more interesting lipids, which are designed to optimize drug delivery or vaccination. Phospho-lipids may still play a role as essential, non-toxic, helper lipids as backbone for the lipid carriers. Therefore, the Phospholipid Research Center will continue its research in this innovative area, hoping that the many interesting and stimulating papers resulting from this research will represent meaningful support of drug delivery.To convert the many exciting research findings into development projects with industrial applications, the PRC will also support feasibility study projects in the future that explore the suitability of certain research findings for further development into products, based on the use of phospholipids.


Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and is spread by arthropod vectors. Virus-like particle (VLP) vaccines, which have the advantages of strong immunogenicity and safety, play an important role in the prevention of this disease. VLPs for RVFV were successfully prepared by our research group using a baculovirus-insect cell expression system. To study the immunogenicity of these RVFV VLPs, a correct 3rd or 4th generation recombinant baculovirus, rBac-N-G, was identified and used to infect Sf9 cells, which were cultured in suspension at a large scale. Subsequently, cell debris was removed by centrifugation, and the VLPs were concentrated by ultracentrifugation and purified using a sucrose gradient, after which they were used to immunize BALB/c mice by intramuscular injection. The results showed that the RVFV VLPs prepared by our research group could effectively induce mice to produce RVFV neutralizing antibodies and that the prepared VLPs could stimulate mouse spleen cells to produce high levels of the cytokines IL-4 and IFN-γ. Moreover, the proportion of lymphocytes producing IL-4 and IFN-γ in the spleen of mice immunized with RVFV VLPs was significantly increased. Therefore, the RVFV VLPs prepared in this study had strong immunogenicity and could effectively activate humoral and cellular immunity in mice. This study lays a solid foundation for the development of RVFV VLP vaccine candidates and promotes the healthy development of animal husbandry and human public health. Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV) that is prevalent in the Arabian Peninsula, African continent and several islands in the southeast Indian Ocean , 2. In tThe National Medium- and Long-term Plan for Animal Disease Control (2012–2020). Due to its significant biological impact, this disease must be prevented and controlled.The US Centers for Disease Control and Prevention (CDC) and the US Department of Agriculture (USDA) list RVFV as a Class A pathogen . China lLaboratory studies have shown that vaccination is the main means of preventing and controlling RVF , 7. No s+ T cells; VLPs are recognized and presented to CD8+ T cells by MHC class I molecules via cross-presentation.VLPs display epitopes in a form close to that of natural viruses, thus rendering them not only highly immunogenic but also easily acquirable by specialized antigen-presenting cells (APCs), i.e., dendritic cells (DCs) . VLPs caIn a previous study, our research group successfully developed a method for the preparation of RVFV VLPs based on an insect baculovirus system . In thisSpodoptera frugiperda Sf9 cells) was preserved by the Laboratory of Animal Virology and Special Animal Lemology at the Military Veterinary Research Institute in China; these cells were cultivated per a previously described method , which was then centrifuged at 30,000 rpm at 4°C for 120 min. Next, the white matter between the 40 and 60% sucrose liquid levels was collected and added to an ultracentrifuge tube with PBS. The resultant solution was centrifuged at 30,000 rpm for 1.5 h, after which the supernatant was discarded, and the pellet was resuspended in PBS and dissolved overnight at 4°C. The solution concentration was determined using a BCA protein assay kit , and other methods were used for verification. The final concentration was adjusted to 1 mg/ml, and the solution was aliquoted and stored at −80°C for further use.A wide host range can be experimentally infected with RVFV, but as mice can be infected by various means, they are a good animal model for studying the pathogenesis of RVFV infection in large mammals. In this experiment, mice were immunized with the purified RVFV VLPs. The mice were randomly divided into 4 groups of 10, with the first group being the PBS group, the second group being the Freund's adjuvant group, the third group being the VLP group and the fourth group being the VLP + Freund's adjuvant group. Freund's complete adjuvant and Freund's incomplete adjuvant were used for the first and second immunization, respectively, with an immunization dose of 100 μl/mouse via hind limb intramuscular injection. Group one was intramuscularly injected with PBS, group two with Freund's adjuvant , group three with VLPs (15 μg VLPs/mouse) and group four with VLPs + Freund's adjuvant (15 μg VLPs/mouse). Blood was collected from the mice at 2 weeks after the second immunization, and immunity-related indexes were determined. The design of the immunization protocol is shown in 450nm value of each sample was determined using a microplate reader  substrate was added and incubated at room temperature for 15 min. The stop solution was added, and the ODe reader .A serum neutralization test was carried out as follows. A previously, constructed RVFV pseudovirus (with an EGFP marker) and neutralization assay  was usedIFN-γ and IL-4 levels were determined using an IFN-γ and IL-4 ELISpot assay kit . At day 14 after the second immunization, spleens were collected from the mice with and without VLPs immunization to isolate spleen cells. All procedures were performed in a biosafety cabinet.+ or CD4+ T cells. Flow cytometry was used to determine cellular fluorescence.Intracellular cytokine staining (ICS) analysis was conducted at day 14 after the second immunization. The mouse spleens with and without VLPs immunization were collected, and spleen cells were isolated for ICS of IL-4 and IFN-γ in CD8t-test was performed to evaluate differences. Significant difference was set at P < 0.05.All data were expressed as the mean ± SD, and statistical analyses were performed using GraphPad Prism 5 software . Student's 7, was produced in the fourth group (the VLPs + Freund's adjuvant group) of mice. The third group (the VLP group) mice also produced a high serum titer, 1:26, in the absence of any adjuvant. The results were shown in The RVFV VLPs immunizations were conducted according to the 7 and 100% neutralization activity at a dilution of 1:26. In sera from mice in the third group , 100% neutralization activity at a dilution of 1:25 and 60% neutralization activity at a dilution of 1:26 were observed. These results showed that VLP-based experimental vaccines have high immunogenicity in mouse models and can induce high levels of neutralizing antibodies even in the absence of adjuvants.The sera prepared from the blood samples taken from the mice on day 14 after the second immunization were doubly diluted, and neutralizing activity was determined using an HIV-based lentiviral-packaged RVFV pseudovirus (with an EGFP marker)  preparedTo determine whether the RVFV VLPs can induce cytokine production in lymphocytes, spleen lymphocytes were isolated at 2 weeks after the booster immunization (week 4). Subsequently, the spot-forming cells (SFCs) produced by the lymphocytes with partial Gn protein stimulation were detected to analyze the ability of the lymphocytes to produce cytokines such as IFN-γ and IL-4. The results are shown in + and CD8+ T cells. The difference between the percentage of cells that were both cytokine-positive and lymphocyte-positive when Gn protein was added and that of cells that were both cytokine-positive and lymphocyte-positive in the absence of Gn protein were graphed for each cytokine, and the results are shown in To effectively assess the intensity of the cellular immune response and to further evaluate the activation of T lymphocytes induced by the RVFV VLPs in mice, spleen cells were isolated at 2 weeks after the booster immunization (week 4), and ICS was used to analyze the synthesis and secretion of IFN-γ and IL-4 in CD4+) isolated from mice in the fourth group (the VLPs + Freund's adjuvant group) were significantly higher than those in the other three groups  from mice in the fourth group (the VLPs + Freund's adjuvant group) produced a significantly higher level of IFN-γ than that of the third group (the VLP group)  play an important role in the fight against viral infection, ICS was used to assess IL-4 and IFN-γ cytokine production in T lymphocytes (CD8+) (+) from mice in the fourth group (the VLPs + Freund's adjuvant group) was significantly higher than that in the other three groups (P < 0.01). Moreover, for lymphocytes from mice in the third group (the VLP group) in which the adjuvant was absent, secreted IFN-γ cytokine production by T lymphocytes (CD8+) was also high. IL-4 secretion by T lymphocytes (CD8+) was similar to that of IFN-γ. The secretion of IL-4 and IFN-γ by T lymphocytes (CD8+) from mice in the first group (the PBS group) and second group (the Freund's adjuvant group) was extremely low.Since T lymphocytes (CD8s (CD8+) . IFN-γ cRVF can be transmitted by mosquito bites and by contact with contaminated materials, and it has a mortality rate of approximately 1%. However, in recent epidemics, the mortality rate has increased significantly . At presVLPs have a polyprotein structure that mimics that of their natural virus in morphology and structure and is free of viral genetic material, thus making them safe and expensive candidate vaccines. At present, VLP-based commercially available preventive vaccines have been developed worldwide , includiIn this study, a previously constructed recombinant baculovirus , rBac-N-+ and CD4+ T lymphocytes secreting IL-4 and IFN-γ in the mouse spleen cells. IFN-γ is a Th1-type cytokine, and IL-4 a Th2-type cytokine, indicating that the VLPs alone can induce a balanced Th1/Th2 immune response in mice. Hence, the prepared RVFV VLPs could induce a strong immune response in small animals. This study showed that VLP-based experimental vaccines (strains) have good immunogenicity in mouse models and can induce high levels of neutralizing antibodies even in the absence of adjuvants.RVFV VLPs have been generated by several other researchers –29, and However, many tasks must be completed to prepare a vaccine with RVFV VLPs using the Bac-to-Bac system. The immunogenicity of the VLPs would be further improved and the production costs would be reduced, thereby laying the foundation for the development of vaccines using RVFV VLPs. In addition, there are some limitations in this study. For example, the virus challenge experiment after vaccine immunization is lacking due to the limited experiment conditions. Further studies are needed for evaluating the vaccine efficiency using animal models.All datasets generated for this study are included in the article/supplementary material.All the animal studies were conducted according to the Guidelines for the Care and Use of Laboratory Animals , and all efforts were made to minimize suffering. The study protocol was approved by the Animal Ethics Committee of the Institute of Military Veterinary Medicine at the Academy of Military Medical Science.YL, SY, and XX designed the experiment. YL, LH, and YZ performed the experiments. XZ, HW, WG, HJ, and GL helped with the experiments. QW, NF, and YG analyzed the experimental results and developed analysis tools. YL wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


After two years of exchange, this specific issue dedicated to the Carnot cycle and thermomechanical engines has been completed with ten papers including this editorial.Our thanks are extended to all the authors for the interesting points of view they have proposed: this issue confirms the strong interactions between fundamentals and applications in thermodynamics.Regarding the list of published papers annexed at the end of this editorial, it appears that six papers ,3,4,5,6 In “OTEC Maximum Net Power Output Using Carnot Cycle and Application to Simplify Heat Exchanger Selection”, Fontaine et al.  considerThe first conclusion is that we must enlarge the subject from engines to reverse cycle machines, and particularly those dedicated to low temperature applications. This is confirmed by the paper “Global Efficiency of Heat Engines and Heat Pumps with Non-Linear Boundary Conditions”  which isTwo papers give respectively a short history regarding efficiency at maximum power and an analysis of the methodology used in modeling and optimization of Carnot engines ,6. It apThe paper by Gonzalez-Ayala et al.  has the The paper by Xue and Guo  is more We hope to have the opportunity to continue to report on the progress of the subject in the near future, extending the subject to reverse cycle configurations.


Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from  Nucleosides triphosphates (NTPs), such as ATP (adenosine triphosphate) and GTP (guanosine triphosphate), are used as energy source or as allosteric effector by a number of proteins, involved for instance, in metabolism, active transport, cell division or DNA/RNA synthesis. However, the mechanism of NTP hydrolysis in proteins are still poorly understood, especially its coupling to functional events, such as movement of proteins along nucleic acids. Indeed, detailed mechanistic insight is lacking for a number of systems, including even intensively studied systems such as dyneins , ABC impHelicobacter pylori involved in DNA replication, and an ABC transporter implicated in multidrug resistance, BmrA (Bacillus subtilis multidrug resistance ATP binding cassette transporter), which share high similarities in their ATP binding sites . . 31P CP To summarize, our data show that analysis of the pre-hydrolytic state is difficult both for DnaB and BmrA, since first the corresponding ATP mimics do not behave in a homogenous manner, i.e., analogues which should yield similar states lead to different NMR spectra, and second, most popular analogues are actually hydrolysed by the helicase in presence of DNA, as well as by the ABC transporter.x for the middle pre-hydrolysis state and AMPPNP for the late pre-hydrolysis state [2 group, a possible hydrogen bond donor; AMPPCP has one oxygen atom less than ATP.With respect to the first point, the intriguing observation that the AMPPCP- and AMPPNP-induced pre-hydrolytic states show conformational differences might be linked to the proposition that one can further differentiate each pre-hydrolytic mimic, as discussed by Ogawa et al., and assign the different mimics to specific steps therein: ATP-γ-S for the initial pre-hydrolysis state, AMPPCP for the pre-isomerization state, ADP:BeFis state . It is d−1·mg−1 one of the most active ABC transporters  [With respect to the second point, in the DnaB-DNA complex, only AMPPCP resisted to hydrolysis, and was the best choice to study DnaB and its DNA complex. It was however, rapidly hydrolysed in BmrA, which might be caused by the very high ATPase activity of BmrA, which is with an activity of 6.5 µmol·minporters) . AmongstIn order to investigate the transition states of BmrA and DnaB, we used the solid-state NMR techniques already described above, and also complemented them by EPR . The con13C-13C 2D DARR spectra of DnaB apo and DnaB:ADP:Vi display a few shifting resonances upon binding of the nucleotide , and foronments  , and the presence of vanadate induces 31P chemical-shift changes for BmrA which were not observed for DnaB [A 1D for DnaB . In casefor DnaB ; BmrA [9for DnaB ; LmrA [1for DnaB ; Maltosefor DnaB ) suggestfor DnaB .51V nucleus (I = 7/2) in proteins in which the Mg2+ has been replaced by the EPR-active Mn2+ metal ion [2+ to the nuclei in the vicinity. We applied this both to the ABC transporter and the DnaB helicase. One should mention that it was shown by biochemical investigations for both proteins that upon substitution of Mg2+ by Mn2+, their biological function is maintained [51V . The presence of the first order quadrupolar coupling sideband pattern already points to immobilized 51V species. We can distinguish two resonances at around −600 ppm (−604 ppm and −618 ppm) and two further vanadate species bound to DnaB at −533 and −681 ppm (51V was recorded and assigned (51V MAS spectrum can be assigned by comparison with the solution-state spectrum of the supernatant as follows: VO43− (V1), V2O74− (V2), V4O124− (V4) and V5O155− (V5) [−1) at pH 6, we are two orders of magnitude below the solubility limit. The detected signal thus must stem from DnaB-bound vanadate, which might be related to the observation that addition of vanadate interferes with DNA binding to DnaB  ,121. We  to DnaB C.13C-13C DARR spectrum, and vanadate is indeed present in the vicinity of the metal ion. In contrast, ADP:Vi is not a suitable ATP-transition-state mimic for the helicase DnaB. Indeed, solid-state NMR and EPR experiments reveal that vanadate does not bind to the NBD together with the nucleotide. Instead, vanadate is bound elsewhere to DnaB, most likely in an unspecific manner. ADP:Vi strongly inhibits binding of DNA, suggesting that they share the same binding site on DnaB, and that vanadate outcompetes DNA.To sum up, vanadate is a reasonable ATP-transition-state mimic for the ABC transporter BmrA. The transporter is trapped, most likely in its outward-facing state, when binding ADP:Vi. The transition state is characterized by a characteristic fingerprint in the NMR x is the most frequently used transition-state analogue 3, which is insoluble. However, the presence of an excess of fluoride shifts the pH upon which Al(OH)3 formation occurs to a higher value. We calculated the concentrations of the different species of aluminium under the conditions used (6 mM of AlCl3 and 30 mM NH4F) as a function of the pH-value 3 at pH ≥ 8. The amount of formed AlFX is thus not sufficient to induce the protein:AlFx complex formation. At the same time, fluorides present in the solution can form a complex with Mg2+ generating the transition-state analogue MgF3−. This effect was followed and confirmed by 19F NMR for the conversion of a protein:ADP:AlF4− complex to a protein:ADP:MgF3− complex by increasing the pH [3− and AlF3 are structurally very similar and some structures comprising AlF3 as transition state mimic are in reality MgF3− because they were obtained at pH ≥ 8 [AlFanalogue A, althoupH-value . In our g the pH . Moreovet pH ≥ 8 ,57,58.x complex can easily be prepared at a pH of 6, since the protein is stable at this pH value. In the presence of the transition-state analogue, the 1D CP 31P spectrum displays two very narrow resonances assigned to the Pα and Pβ of ADP in complex with AlFx  spectra. Nevertheless, we explored this further, and in order to test the pH dependency, BmrA, in the presence of ATP, was incubated with 6 mM of AlCl3 and 30 mM NH4F at pH 8, 7.5 and 7, and a 1D CP 31P NMR was recorded for all three conditions (31P NMR spectrum at pH 8 shows that ATP/ADP is abundantly co-precipitated with Al(OH)3 which makes the 1D spectrum difficult to analyse due to a broad and rather unstructured resonance of this amorphous species 3 is still visible in the 31P NMR spectrum, one can compare it to BmrA:ADP:Vi, which shows that both spectra overlay with only few minor differences 3 at pH ≥ 7 under our conditions. The spectra of BmrA at pH ≥ 7 well illustrate the consequences of the use of AlFx in alkaline conditions. The protein actually adopts a similar conformation as in the presence of vanadate, but high amounts of amorphous species are detected. In contrast, DnaB at pH 6 shows high affinity to AlFx which induces substantial conformational changes; and also DNA binding is not affected.To summarize, the use of AlF31P and 13C experiments described above to characterize BmrA and DnaB in the presence of ADP.The last state in the ATP hydrolysis cycle is the post-hydrolytic state, where ADP is still bound to the protein and the inorganic phosphate (previously γ-phosphate) is released from the binding pocket. This state is well mimicked by the addition of ADP. We used the 31P CP spectrum for DnaB:ADP is shown in N-terminal domain peaks, indicating conformational changes and an increase in the dynamics of the protein.The 31P CP spectrum shows the presence of two populations of ADP as identified by peak doubling, labeled Pα, Pβ, and Pα’, Pβ’  in the outward-facing conformation, with two interacting NBDs and in the presence of AMPPNP (PDB 3RLF), ADP:BeF3 (PDB 3PUX), ADP:Vi (PDB 3PUV), and ADP:AlF4- (PDB 3PUW) [4−) are (i) the distance between the γ-phosphate or the mimicked γ-phosphate by the analogues and the bridging oxygen of the β-phosphate and (ii) the presence of a water molecule, essential for the ATP hydrolysis, only in the transition state. Although the transmembrane part of the maltose transporter essentially differs from that of BmrA, the NBD homodimers of the two proteins are relatively similar . The two NBDs differ mainly in their ATPase activity. The ATPase activity of MBP-MalFGK2 is one order of magnitude lower than BmrA [4− might be taken as reporters of the enzyme transition state; in the presence of AlF4−, the crystal structure of the maltose transporter shows a classical picture with the catalytic water molecule in the apical attack position  . The colDB 3PUW) . Chen anhan BmrA . For Bmrhan BmrA  and NMR han BmrA . It is bposition A. Even iosphates D, respecDnaB of Helicobacter pylori and its structural counterparts. The conformation of DnaB, as could be judged from the 2D 13C-13C DARR spectra, essentially depends on the nature of the analogue used, which matches the great structural variability reported for DnaB from other bacteria and their viral homologues [Bacillus stearothermophilus (currently Geobacillus stearothermophilus), which was crystalized, in the presence of a DNA strand, with GDP:AlF4− in five of its six catalytic sites [4− in the structure of Geobacillus stearothermophilus DnaB  to provide information at the atomic level on the binding of mimics of the different ATP forms present during the hydrolysis cycle. We investigated this for two ATP-fuelled proteins, an ABC transporter and a DNA helicase , both dr


Heart failure with preserved ejection fraction (HFpEF) is a complex heterogeneous disease for which our pathophysiological understanding is still limited and specific prevention and treatment strategies are lacking. HFpEF is characterised by diastolic dysfunction and cardiac remodelling . Recently, microvascular dysfunction and chronic low-grade inflammation have been proposed to participate in HFpEF development. Furthermore, several recent studies demonstrated the occurrence of generalized lymphatic dysfunction in experimental models of risk factors for HFpEF, including obesity, hypercholesterolaemia, type 2 diabetes mellitus (T2DM), hypertension, and aging. Here, we review the evidence for a combined role of coronary (micro)vascular dysfunction and lymphatic vessel alterations in mediating key pathological steps in HFpEF, including reduced cardiac perfusion, chronic low-grade inflammation, and myocardial oedema, and their impact on cardiac metabolic alterations , fibrosis, and cardiomyocyte stiffness. We focus primarily on HFpEF caused by metabolic risk factors, such as obesity, T2DM, hypertension, and aging. More than half of the patients with heart failure (HF), notably women, suffer from HF with preserved ejection fraction , a complex cardiovascular syndrome characterised by diastolic dysfunction and cardiac stiffening, fibrosis, inflammation, and hypertrophy. As a consequence of population aging, as well as the increases in common comorbidities, such as obesity, type 2 diabetes mellitus (T2DM), and hypertension, the prevalence of HFpEF is rising . Alarminmicrovascular paradigm proposes endothelial dysfunction as the central mediator connecting chronic systemic low-grade inflammation with myocardial dysfunction and remodelling in HFpEF  and constrictors (e.g. endothelin 1) . En. En34]. ath Fig.  52]. In. In2−)-mts Table  35]. Be. Be2−)-mN-acetylcysteine (NAC) prevented diastolic dysfunction in rodent models of diabetes and hypertensive cardiomyopathy [In HF patients and experimental mouse models, several anti-oxidative stress therapies have been investigated that either (1) inhibit oxidative stress producers, (2) improve endogenous antioxidant capacity, or (3) supplement exogeneous antioxidants. Mitochondria Szeto-Schiller-31  attenuated cardiac remodelling in hypertensive cardiomyopathy and is currently investigated as a novel therapeutic in phase II trials for HFpEF (NCT02814097). Furthermore, treatment with the mitochondria-targeted antioxidant MitoTEMPO or myopathy , 130. Sumyopathy . In summmicrovascular wall barrier dysfunction in HFpEF-associated comorbidities [The microvascular endothelium is a barrier opposing free exchange between blood and tissues, which tightly regulates transport of plasma constituents and immune cells in most organs. Events such as ischaemia cause increased vascular endothelial growth factor (VEGF)-A levels, leading to vascular barrier breakdown, increased extravasation of immune cells, and oedema. In addition to its well-known occurrence in HFrEF, there is accumulating evidence of bidities , 79, 128endothelial glycocalyx covers the luminal surface of vascular endothelial cells and together with cell–cell junctions serves as a barrier for solute and macromolecule exchanges. It is also a mechanotransducer, which senses endothelial shear stress, attenuates coagulation and leucocyte adhesion to the endothelium, and affects vasoregulatory responses to flow. A damaged glycocalyx induces the production of pro-adhesion mediators, thereby triggering the adherence of neutrophils to the endothelium  is another proposed route of transcytosis-mediated extravasation during vascular leakage  foci, while vascular pericyte coverage was reduced compared to controls [Beyond endothelial cells, controls . FurtherThe heart has an extensive lymphatic network that relies on cardiac contractions to propel lymph fluid towards draining cardiac lymph nodes . CardiacLymphatic dysfunction and remodelling has been shown to occur in experimental models of multiple HFpEF-associated comorbidities  and cardiomyocyte passive stiffness (titin modifications) reduce LV compliance, as observed in HFpEF patients .2+/calmodulin-dependent kinase-II (CAMKII) also modify passive stiffness [11878 and reduced phosphorylation on N2B unique sequence (N2Bus) S4185, which was associated with increased LV stiffness [The giant sarcomeric protein titin, a force-transducing bidirectional spring, is the main determinant of cardiomyocyte stiffness. Alternative splicing of titin mRNA creates isoforms with differential stiffnesses: the short, stiffer N2B isoform, and the longer, more compliant N2BA isoform. Extension of the elastic I-band region in both titin isoforms supports myocardial passive relaxation during diastole. Post-translational modifications of titin rapidly alter cardiomyocyte stiffness. In HFpEF patients, hypophosphorylation of N2B and ex vivo administration of PKG are associated with increased LV stiffness , 125. Intiffness . HFpEF ptiffness . In additiffness . Howevertiffness . AltogetMyocardial fibrosis and extracellular matrix accumulation are hallmarks of adverse cardiac remodelling associated with HFpEF and its comorbidities Table  58, 84,, 136. ThE/E′) in HFpEF patients [Lysyl oxidases (LOXs) are the main enzymes involved in collagen and elastin cross-linking during cardiac remodelling . Upregulpatients . Furtherpatients . Cardiacpatients . Pro-infpatients . The rolpatients .Several anti-fibrotic therapeutics, focused on inhibiting aldosterone signalling with mineralocorticoid receptor antagonists (MRA), have been trialled in patients with in HFpEF . PromisiThe healthy heart requires a large, constant supply of energy of which 30% is provided by carbohydrate oxidation (mainly glucose and lactate) and 70% by beta-oxidation of free fatty acids (FFAs) at rest. Cardiac metabolic alterations in HFpEF patients have been poorly investigated and current hypotheses are largely based on metabolic alterations observed in the separate comorbidities Table . For exaInterventions, such as caloric restriction, and insulin-sensitizing or glucose-lowering agents, such as metformin, dipeptidyl peptidase-4 (DPP-4) inhibitors, and glucagon-like peptide-1 (GLP-1) receptor agonists, all improve murine diastolic function . In HFrECurrently, effective and specific therapies for HFpEF are lacking due to incomplete understanding of disease mechanisms. Advances in this area are hindered in part by the limited number of adequate animal models. Indeed, the disease mechanisms that were discovered in experimental animal studies may be influenced by the short-comings to account for comorbidities, age, gender, and hormonal status of current experimental animal models. Given that HFpEF is such a clinically heterogeneous disease, there may not be one single uniting pathological mechanism driving the disease and, therefore, having multiple pre-clinical animal models that can be compared to human pathophysiology is critical. Due to their similarity to human disease progression, large animal models, such as the multiple comorbidity swine model , are espSymptoms of HFpEF are not disease-specific and are often difficult to interpret in patients, leading to inadequate or late diagnosis , 44, 107Nevertheless, it is envisaged that therapies targeting generalized vascular dysfunction, such as (1) anti-inflammatory drugs, (2) antioxidants, (3) anti-vascular permeability drugs , and/or (4) anti-vascular rarefaction drugs (such as angiogenic therapy) might prevent or treat HFpEF by restoring cardiac perfusion and attenuating cardiac inflammation. The translation of vascular drugs to clinical practice in HFpEF, as well as HFrEF, is still difficult due to the complex and multifactorial pathophysiology of the coronary circulation , 68. In Notably, as HFpEF is a multifactorial disease, combined personalized medicine targeting cardiac vascular and lymphatic dysfunction, fibrosis, inflammation, and metabolic flux alterations may be needed to limit diastolic dysfunction and improve quality of life.


Mulberry mosaic dwarf-associated virus (MMDaV), an unsigned species of the family Geminiviridae. We show that the V2 protein colocalizes with the nucleolar protein fibrillarin (NbFib2) in the nucleolus upon transient expression in the epidermal cells of Nicotiana benthamiana. A yeast-two hybrid assay, followed by bimolecular fluorescence complementation assays, demonstrated the specific interaction between V2 and NbFib2. Intriguingly, we find that the presence of MMDaV excludes the V2 protein from the nucleolus to nucleoplasm. We present evidence that the replication-associated protein A (RepA) protein of MMDaV interacts with V2 and enables the nucleolar exclusion of V2. We also show that, while V2 interacts with itself primarily in the nucleolus, the presence of RepA redirects the site of V2–V2 interaction from the nucleolus to the nucleoplasm. We further reveal that RepA promotes V2 out of the nucleolus presumably by directing the NbFib2-V2 complex from the nucleolus to the nucleoplasm. Considering the critical role of the nucleolus in plant virus infection, this RepA-dependent modulation of V2 nucleolar localization would be crucial for understanding the involvement of this subcellular compartment in plant–virus interactions.Plant viruses have limited coding capacities so that they rely heavily on the expression of multifunctional viral proteins to achieve a successful infection. The functional specification of viral proteins is often related to their differential interaction with plant and viral components and somewhat depends on their localization to various subcellular compartments. In this study, we analyzed the intracellular localization of the V2 protein of  With limited coding capacities, plant viruses usually encode multifunctional viral proteins that play important roles in several steps of a virus life cycle. The functional specification of these multifunctional viral proteins is often related to their differential interaction with host plant and viral components and somewhat depends on their dynamic localization to various subcellular compartments . UnderstThe plant nucleolus is the prominent subnuclear structure of cells that comprises several functional subcompartments such as fibrillar centers, a dense fibrillary component, a granular component, nucleolar vacuoles, nucleonema, and nucleolar chromatin . BesidesBecurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus  viruses that replicate in the nucleus. Based on their genome organization, host range, and insect vector, geminiviruses have been classified into nine genera, namely, rtovirus . The genrtovirus . These drtovirus . Assemblrtovirus . Due to rtovirus . As the rtovirus . Reminisrtovirus  and dsDNrtovirus , CP of artovirus . This dyCP gene, is present in the virion-sense strand of different geminiviruses but not in members of the New World bipartite begomoviruses. The V2/AV2 protein of several geminiviruses has been identified as a suppressor of posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS)  . V2/AV2 ng (TGS) . Althougng (TGS) , the subng (TGS) . TYLCV Vng (TGS) , and to ng (TGS) . The AV2ng (TGS) . Grapeving (TGS) . The V2 a plants . Most ofa plants , it is pN. benthamiana in the absence or presence of MMDaV infection. We show that the MMDaV V2 protein colocalizes with the nucleolar protein fibrillarin in the nucleolus upon transient expression of V2 in the epidermal cells of N. benthamiana. Strikingly, we find that the V2 protein is excluded from the nucleolus to nucleoplasm in the context of MMDaV infection. We also demonstrate that the RepA interacts with V2 and mediates the change of V2 subcellular localization.In this study, we analyzed the dynamic subcellular localization of MMDaV V2 in N. benthamiana and transgenic N. benthamiana plants expressing a red fluorescent protein (RFP)-tagged histone 2B nuclear marker protein (RFP-H2B plants) (Wild-type  plants)  were groRepA, V2, and V3 genes from MMDaV (GenBank accession number KP303687)  were cloned into pENTR-D/TOPO entry vector without stop codon by standard protocols  to yield pENTR-RepA, pENTR-V2, pENTR-V3, pENTR-V2dm61–77aa, and pENTR-NbFib2, respectively. For subcellular localization analysis, NbFib2 and RepA was gateway-cloned into plant binary vectors pEarleyGate102 [N-terminal cyan fluorescent protein (CFP) fusion] and pGWB454  to yield Fib2-RFP and RepA-CFP, respectively  were constructed previously , respectively. The oligonucleotide primers used to generate the plasmids are listed in The P303687) , the V2dm61–77aa  and the ectively . The expeviously . To prodeviously . To geneV1, V3, V4, V5, RepA, and Rep) were constructed in a previous study . The recombinant plant binary pCHF3 vectors expressing the individual ORFs of MMDaV , and 100 μM acetosyringone, and infiltrated into 4-week-old wild-type N. benthamiana or RFP-H2B transgenic N. benthamiana leaves as described  at 36–48 hours post-infiltration (hpi). Leaf tissues were examined using a Zeiss c-Apochromat 40 × 1.2 water immersion Korr objective. The GFP fluorophore was excited with 488-nm laser lines and emission was detected at 500–550 nm. To avoid chlorophyll auto-fluorescence, the RFP fluorophore was excited with 561-nm laser lines and emission was detected at 600–630 nm. The YFP fluorophore was excited with 514-nm laser lines and emission was detected at 519–587 nm. The CFP fluorophore was excited with 458-nm laser lines and emission was detected at 470–500 nm. For every experimental sample, at least three independent biological replicates were examined. Images were processed with Zeiss ZEN software.Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated Total RNA was extracted from infiltrated leaf regions of RFP-H2B plants using TRIzol reagent  following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using PrimeScript RT reagent Kit with genomic DNA Eraser . Expression of the individual ORF of MMDaV was detected by reverse transcription PCR (RT-PCR) from the synthesized cDNA as previously described .Total plant proteins were extracted from infiltrated leave patches as described . SupernaGAL4-based yeast two-hybrid (Y2H) assays were carried out following the Clontech yeast protocol handbook. In brief, both plasmids containing fusion proteins with the GAL4 activation domain and DNA-binding domain were cotransformed into the yeast strain Y2H Gold and plated onto a selective medium lacking tryptophan and leucine (SD/-Trp/-Leu) at 30°C for 3–5 days to select for doubly transformed yeast cells. Transformants were then transferred to the selective medium lacking leucine, histidine, and tryptophan (SD/-Leu/-His/-Trp) containing 5 mM 3-amino-1,2,4-triazole (3-AT) or the selective medium lacking adenine, histidine, leucine and tryptophan (SD/-Ade/-His/-Leu/-Trp) and cultured at 30°C for 3–5 days to identify possible interaction by activation of reporter genes His3 and/or Trp.N. benthamiana plants expressing RFP-H2B (hereafter designated as RFP-H2B plants). These plants contain a 2 × 35 Spro:RFP-H2B expression cassette which allow us to monitor the nucleoplasm but not the nucleolus of plant cells (n = 60)  . Despite(n = 60) . This wa(n = 60) .N. benthamiana plant leaves expressing NbFib2-RFP with GFP showed that NbFib2 localized to the nucleolus and Cajal body of the epidermal cells of N. benthamiana plant leaves , and occasionally in some structures around/outside the nucleus in about 33% of the cells  pGADT7 and “bait” pGBKT7 vectors to produce AD and BD fusions, and the bait and prey plasmids were then cotransformed to yeast strain Y2H Gold. Cotransformation of murine tumor suppressor p53 and simian virus 40 large T antigen (T) was used as a positive control and that of human lamin (Lam) and T as a negative control. Similar to the positive control, robust growth of yeast transformants containing BD-NbFib2 and AD-V2 was observed on the selective medium lacking histidine, leucine, and tryptophan supplementing with 5 mM 3-AT, whereas yeast transformants harboring AD-V2 with the empty BD vector or BD-NbFib2 with the empty AD vector failed to grow on the selective triple dropout medium, suggesting the specific interaction between NbFib2 and V2 in yeast cells . NbFib2-he cells , indicatSince mutation of the predicted nuclear localization signal  of MMDaV V2 abolished its subnuclear foci localization and PTGS suppression activity , we perfn = 60). However, coexpression of GFP with MMDaV did not cause any profound shift in the localization of GFP  . For exain planta was only observed in the nucleoplasm by the BiFC combination of RepA and V2   , suggestN. benthamiana plant leaves with MMDaV, NbFib2-nYFP, and V2-cYFP  construct was generated and coexpressed with the BiFC combination of V2 and NbFib2. Interestingly, when RepA-CFP was coexpressed with NbFib2-nYFP and V2-cYFP in RFP-H2B plant leaves, an altered pattern of BiFC signal distribution was detected in infiltrated cells. Coexpression of the RepA-CFP protein with NbFib2-nYFP and V2-cYFP consequently redistributed the V2-NbFib2 complex out of the nucleolus to the nucleoplasm . Exclusi V2-cYFP . These dN. benthamiana plants, V2 is excluded from the nucleolus in cells infected with MMDaV. This localization difference of V2 is associated to the presence of the RepA protein of MMDaV.Geminiviruses are known to have a limited coding capacity and have compensated for this restriction by encoding multifunctional viral proteins to make use of host biosynthetic machinery and to suppress multiple layers of plant immune responses to achieve a successful infection . PerhapsViral proteins targeting the nucleus generally rely on a nuclear localization signal consisting of a short stretch of basic residues that allows interaction with the nuclear import proteins, importins, mediating entry into the nucleus via the nuclear pore complex . In contBesides its accumulation in the nucleolus, MMDaV V2 also targets to other subnuclear foci outside of the context of virus infection. Note that the numbers of the subnuclear foci are approximations as it is difficult to assess due to their unclear boundaries and aggregation in the nucleus in some cases . InteresStrikingly, V2 is excluded from the nucleolus to the nucleoplasm in plant cells infected with MMDaV. Although this virus-dependent exclusion of V2 from the nucleolus might reflect more the natural situation in the viral infection process, we cannot rule out the possibility of temporal and spatial distribution of V2 to the nucleolus. Transporting of viral protein to different subcellular compartments has also been implicated to play critical roles in functional specification of plant viruses. For example, the nuclear-cytoplasmic compartmentalization of cucumber mosaic virus (CMV) 2b presumably maximizes the benefit for CMV by regulating the balance between virus accumulation and damage to the host . NucleocWe also presented evidence that the dynamic localization of V2 and V2–V2 self-interaction site is RepA-dependent. We found that RepA redirects the V2-NbFib2 complex from the nucleolus to the nucleoplasmic sites. As RepA is involved in geminivirus replication, RepA-V2 interaction-mediated nucleolar exclusion of V2 might imply a link between virus replication and silencing suppression. Recruitment of the BSMV γb protein by the αa viral replication protein to the chloroplast membrane site of BSMV replication enhances the helicase activities of αa in unwinding of dsRNA duplexes and promotes BSMV replication . It needWe have shown that MMDaV V2 displays a differential subcellular localization pattern in response to MMDaV infection. We also show that RepA interacts with V2 and mediates the nucleolar exclusion of V2. As many different viruses target the nucleolus to aid virus infection, the study of the interaction of viral proteins with the nucleolus will provide insight into the mechanisms of viral manipulating host cell functions which could lead to the design of novel antiviral strategies.All datasets generated for this study are included in the article/DW, SS, YR, and XY performed the experiments. All the authors contributed to the experimental design and interpretation. XY, DW, and XZ wrote the manuscript with contributions from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.