Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite.

The inner membrane complex (IMC) is a unifying morphological feature of all alveolate organisms. It consists of flattened vesicles underlying the plasma membrane and is interconnected with the cytoskeleton. Depending on the ecological niche of the organisms, the function of the IMC ranges from a fundamental role as reinforcement system to more specialized roles in motility and cytokinesis. In this article, we present a comprehensive evolutionary analysis of IMC components, which exemplifies the adaptive nature of the IMCs' protein composition. Focusing on eight structurally distinct proteins in the most prominent "genus" of the Alveolata-the malaria parasite Plasmodium-we demonstrate that the level of conservation is reflected in phenotypic characteristics, accentuated in differential spatial-temporal patterns of these proteins in the motile stages of the parasite's life cycle. Colocalization studies with the centromere and the spindle apparatus reveal their discriminative biogenesis. We also reveal that the IMC is an essential structural compartment for the development of the sexual stages of Plasmodium, as it seems to drive the morphological changes of the parasite during the long and multistaged process of sexual differentiation. We further found a Plasmodium-specific IMC membrane matrix protein that highlights transversal structures in gametocytes, which could represent a genus-specific structural innovation required by Plasmodium. We conclude that the IMC has an additional role during sexual development supporting morphogenesis of the cell, which in addition to its functions in the asexual stages highlights the multifunctional nature of the IMC in the Plasmodium life cycle.

Sequence requirements for the export of the Plasmodium falciparum Maurer's clefts protein REX2.

A short motif termed Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS) characterizes Plasmodium proteins exported into the host cell. These proteins mediate host cell modifications essential for parasite survival and virulence. However, several PEXEL-negative exported proteins indicate that the currently predicted malaria exportome is not complete and it is unknown whether and how these proteins relate to PEXEL-positive export. Here we show that the N-terminal 10 amino acids of the PEXEL-negative exported protein REX2 (ring-exported protein 2) are necessary for its targeting and that a single-point mutation in this region abolishes export. Furthermore we show that the REX2 transmembrane domain is also essential for export and that together with the N-terminal region it is sufficient to promote export of another protein. An N-terminal region and the transmembrane domain of the unrelated PEXEL-negative exported protein SBP1 (skeleton-binding protein 1) can functionally replace the corresponding regions in REX2, suggesting that these sequence features are also present in other PEXEL-negative exported proteins. Similar to PEXEL proteins we find that REX2 is processed, but in contrast, detect no evidence for N-terminal acetylation.

GLP: A requirement in cell therapies - perspectives for the cardiovascular field.

In biomedical research, enormous progress is being made and new candidates for putative medicinal products emerge. However, most published preclinical data are not conducted according to the standard Good Laboratory Practice (GLP). GLP is mandatory for preclinical analysis of Advanced Therapy Medicinal Products (ATMP) and thereby a prerequisite for planning and conduction of clinical trials. Not inconsiderable numbers of clinical trials are terminated earlier or fail - do inadequate testing strategies or missing specialized assays during the preclinical development contribute to this severe complex of problems? Unfortunately, there is also a lack of access to GLP testing results and OECD (Organisation for Economic Co-operation and Development) GLP guidelines are not yet adjusted to ATMP specialties. Ultimately, GLP offers possibilities to generate reliable and reproducible data. Therefore, this review elucidates different GLP aspects in drug development, speculates on reasons of putative low GLP acceptance in the scientific community and mentions solution proposals.

Stem cells and heart disease - Brake or accelerator?

After two decades of intensive research and attempts of clinical translation, stem cell based therapies for cardiac diseases are not getting closer to clinical success. This review tries to unravel the obstacles and focuses on underlying mechanisms as the target for regenerative therapies. At present, the principal outcome in clinical therapy does not reflect experimental evidence. It seems that the scientific obstacle is a lack of integration of knowledge from tissue repair and disease mechanisms. Recent insights from clinical trials delineate mechanisms of stem cell dysfunction and gene defects in repair mechanisms as cause of atherosclerosis and heart disease. These findings require a redirection of current practice of stem cell therapy and a reset using more detailed analysis of stem cell function interfering with disease mechanisms. To accelerate scientific development the authors suggest intensifying unified computational data analysis and shared data knowledge by using open-access data platforms.

GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration.

BACKGROUND: CD133+ stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133+ stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics. METHODS: CD133+ stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133+ cells was evaluated and compared to manually isolated CD133+ cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction. RESULTS: We established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 106 viable CD133+ cells with a mean log10 depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo. CONCLUSIONS: Our results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133+ CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs.

Cardiac Function Improvement and Bone Marrow Response -: Outcome Analysis of the Randomized PERFECT Phase III Clinical Trial of Intramyocardial CD133+ Application After Myocardial Infarction.

OBJECTIVE: The phase III clinical trial PERFECT was designed to assess clinical safety and efficacy of intramyocardial CD133+ bone marrow stem cell treatment combined with CABG for induction of cardiac repair. DESIGN: Multicentre, double-blinded, randomised placebo controlled trial. SETTING: The study was conducted across six centres in Germany October 2009 through March 2016 and stopped due slow recruitment after positive interim analysis in March 2015. PARTICIPANTS: Post-infarction patients with chronic ischemia and reduced LVEF (25-50%). INTERVENTIONS: Eighty-two patients were randomised to two groups receiving intramyocardial application of 5ml placebo or a suspension of 0.5-5×106 CD133+. OUTCOME: Primary endpoint was delta (∆) LVEF at 180days (d) compared to baseline measured in MRI. FINDINGS (PRESPECIFIED): Safety (n=77): 180d survival was 100%, MACE n=2, SAE n=49, without difference between placebo and CD133+. Efficacy (n=58): The LVEF improved from baseline LVEF 33.5% by +9.6% at 180d, p=0.001 (n=58). Treatment groups were not different in ∆LVEF (ANCOVA: Placebo +8.8% vs. CD133+ +10.4%, ∆CD133+vs placebo +2.6%, p=0.4). FINDINGS (POST HOC): Responders (R) classified by ∆LVEF≥5% after 180d were 60% of the patients (35/58) in both treatment groups. ∆LVEF in ANCOVA was +17.1% in (R) vs. non-responders (NR) (∆LVEF 0%, n=23). NR were characterized by a preoperative response signature in peripheral blood with reduced CD133+ EPC (RvsNR: p=0.005) and thrombocytes (p=0.004) in contrast to increased Erythropoeitin (p=0.02), and SH2B3 mRNA expression (p=0.073). Actuarial computed mean survival time was 76.9±3.32months (R) vs. +72.3±5.0months (NR), HR 0.3 [Cl 0.07-1.2]; p=0.067.Using a machine learning 20 biomarker response parameters were identified allowing preoperative discrimination with an accuracy of 80% (R) and 84% (NR) after 10-fold cross-validation. INTERPRETATION: The PERFECT trial analysis demonstrates that the regulation of induced cardiac repair is linked to the circulating pool of CD133+ EPC and thrombocytes, associated with SH2B3 gene expression. Based on these findings, responders to cardiac functional improvement may be identified by a peripheral blood biomarker signature. TRIAL REGISTRATION: ClinicalTrials.govNCT00950274.

Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites.

Central to the pathogenesis of malaria is the proliferation of Plasmodium falciparum parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and host cell. One key ligand, Apical Membrane Antigen 1 (AMA1), is a leading blood-stage vaccine and previous work indicates that phosphorylation of its cytoplasmic domain (CPD) is important to its function during invasion. Here we investigate the significance of each of the six available phospho-sites in the CPD. We confirm that the cyclic AMP/protein kinase A (PKA) signalling pathway elicits a phospho-priming step upon serine 610 (S610), which enables subsequent phosphorylation in vitro of a conserved, downstream threonine residue (T613) by glycogen synthase kinase 3 (GSK3). Both phosphorylation steps are required for AMA1 to function efficiently during invasion. This provides the first evidence that the functions of key invasion ligands of the malaria parasite are regulated by sequential phosphorylation steps.

Plasmodium falciparum ATG8 implicated in both autophagy and apicoplast formation.

Amino acid utilization is important for the growth of the erythrocytic stages of the human malaria parasite Plasmodium falciparum, however the molecular mechanism that permits survival of the parasite during conditions of limiting amino acid supply is poorly understood. We provide data here suggesting that an autophagy pathway functions in P. falciparum despite the absence of a typical lysosome for digestion of the autophagosomes. It involves PfATG8, which has a C-terminal glycine which is absolutely required for association of the protein with autophagosomes. Amino acid starvation provoked increased colocalization between PfATG8- and PfRAB7-labeled vesicles and acidification of the colabeled structures consistent with PfRAB7-mediated maturation of PfATG8-positive autophagosomes; this is a rapid process facilitating parasite survival. Immuno-electron microscopic analyses detected PfRAB7 and PfATG8 on double-membrane-bound vesicles and also near or within the parasite's food vacuole, consistent with autophagosomes fusing with the endosomal system before being routed to the food vacuole for digestion. In nonstarved parasites, PfATG8, but not PfRAB7, was found on the intact apicoplast membrane and on apicoplast-targeted vesicles and apicoplast remnants when the formation of the organelle was disrupted; a localization also requiring the C-terminal glycine. These findings suggest that in addition to a classical role in autophagy, which involves the PfRAB7-endosomal system and food vacuole, PfATG8 is associated with apicoplast-targeted vesicles and the mature apicoplast, and as such contributes to apicoplast formation and maintenance. Thus, PfATG8 may be unique in having such a second role in addition to the formation of autophagosomes required for classical autophagy.