Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite.

Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony-is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission.

Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing.

BACKGROUND AIMS: The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX®. METHODS: Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. RESULTS: Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. DISCUSSION: This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products.

Self-assembled 3D spheroids and hollow-fibre bioreactors improve MSC-derived hepatocyte-like cell maturation in vitro.

3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.

Genetic crosses and complementation reveal essential functions for the Plasmodium stage-specific actin2 in sporogonic development.

Malaria parasites have two actin isoforms, ubiquitous actin1 and specialized actin2. Actin2 is essential for late male gametogenesis, prior to egress from the host erythrocyte. Here, we examined whether the two actins fulfil overlapping functions in Plasmodium berghei. Replacement of actin2 with actin1 resulted in partial complementation of the defects in male gametogenesis and, thus, viable ookinetes were formed, able to invade the midgut epithelium and develop into oocysts. However, these remained small and their DNA was undetectable at day 8 after infection. As a consequence sporogony did not occur, resulting in a complete block of parasite transmission. Furthermore, we show that expression of actin2 is tightly controlled in female stages. The actin2 transcript is translationally repressed in female gametocytes, but translated in female gametes. The protein persists until mature ookinetes; this expression is strictly dependent on the maternally derived expression. Genetic crosses revealed that actin2 functions at an early stage of ookinete formation and that parasites lacking actin2 are unable to undergo sporogony in the mosquito midgut. Our results provide insights into the specialized role of actin2 in Plasmodium development in the mosquito and suggest that the two actin isoforms have distinct biological functions.

Equine Musculoskeletal Pathologies: Clinical Approaches and Therapeutical Perspectives-A Review.

Musculoskeletal injuries such as equine osteoarthritis, osteoarticular defects, tendonitis/desmitis, and muscular disorders are prevalent among sport horses, with a fair prognosis for returning to exercise or previous performance levels. The field of equine medicine has witnessed rapid and fruitful development, resulting in a diverse range of therapeutic options for musculoskeletal problems. Staying abreast of these advancements can be challenging, prompting the need for a comprehensive review of commonly used and recent treatments. The aim is to compile current therapeutic options for managing these injuries, spanning from simple to complex physiotherapy techniques, conservative treatments including steroidal and non-steroidal anti-inflammatory drugs, hyaluronic acid, polysulfated glycosaminoglycans, pentosan polysulfate, and polyacrylamides, to promising regenerative therapies such as hemoderivatives and stem cell-based therapies. Each therapeutic modality is scrutinized for its benefits, limitations, and potential synergistic actions to facilitate their most effective application for the intended healing/regeneration of the injured tissue/organ and subsequent patient recovery. While stem cell-based therapies have emerged as particularly promising for equine musculoskeletal injuries, a multidisciplinary approach is underscored throughout the discussion, emphasizing the importance of considering various therapeutic modalities in tandem.

The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis.

BACKGROUND: ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. METHODS: UCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. RESULTS: UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations. CONCLUSION: The results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Corrigendum: Consistent long-term therapeutic efficacy of human umbilical cord matrix-derived mesenchymal stromal cells after myocardial infarction despite individual differences and transient engraftment.

[This corrects the article DOI: 10.3389/fcell.2021.624601.].

Expanding ASM models towards integrated processes for short-cut nitrogen removal and bioplastic recovery.

In next-generation water resource recovery facilities (WRRFs), it is becoming increasingly important to save energy costs and promote resource recovery of valuable products. One way of reducing the substantial aeration energy costs at WRRFs is to employ shortcut N removal, while polyhydroxyalkanoate (PHA) production and recovery as bioplastic is a promising means of recovering a valuable product from biosolids. Both objectives can be achieved simultaneously through the Short-Cut Enhanced Phosphorus and PHA Recovery (SCEPPHAR) process. However, current mathematical models have not previously been employed to describe the behavior of such a process, which limits engineering design and optimisation of process operation. This work focusses on extending the ASM3 model towards the description of short-cut nitrogen removal and simultaneous PHA recovery in a sidestream treatment process. The calibrated and validated model described very well the nitritation process coupled with the aerobic feast/anoxic famine process for the selection of PHA producing organisms at a pilot-scale facility operated in Carbonera, Italy, where the normalised root mean squared error (NRMSE) was consistently <20%. Furthermore, the model applied to the PHA selection stage could effectively describe the PHA accumulation stage without recalibration. A simulation study was performed using the modified ASM3 model to assess the relative benefits of the SCEPPHAR process strategy as compared to the fully aerobic selection process for mixed culture PHA production. While the level of PHA production was found to be 34% lower with SCEPPHAR, a 43% savings in volatile fatty acids (VFAs) demand, a 15% decrease in Total suspended solids (TSS) production and a 28% decrease in oxygen demand were also achievable, which could lead to savings in operational costs. This study facilitates the design and optimisation of WRRFs that integrate short-cut N removal with PHA production, saving aeration energy costs while achieving resource recovery.

3D-MSCs A151 ODN-loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution.

INTRODUCTION: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. OBJECTIVES: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. METHODS: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. RESULTS: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-ß, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-ß, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. CONCLUSION: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines.

Gliding motility protein LIMP promotes optimal mosquito midgut traversal and infection by Plasmodium berghei.

Substrate-dependent gliding motility is key to malaria transmission. It mediates host cell traversal, invasion and infection by Plasmodium and related apicomplexan parasites. The 110 amino acid-long cell surface protein LIMP is essential for P. berghei sporozoites where it is required for the invasion of the mosquito's salivary glands and the liver cells of the rodent host. Here we define an additional role for LIMP during mosquito invasion by the ookinete. limp mRNA is provided as a translationally repressed mRNP (messenger ribonucleoprotein) by the female gametocyte and the protein translated in the ookinete. Parasites depleted of limp (Δlimp) develop ookinetes with apparent normal morphology and no defect during in vitro gliding motility, and yet display a pronounced reduction in oocyst numbers; compared to wildtype 82 % more Δlimp ookinetes remain within the mosquito blood meal explaining the decrease in oocysts. As in the sporozoite, LIMP exerts a profound role on ookinete infection of the mosquito.

Consistent Long-Term Therapeutic Efficacy of Human Umbilical Cord Matrix-Derived Mesenchymal Stromal Cells After Myocardial Infarction Despite Individual Differences and Transient Engraftment.

Human mesenchymal stem cells gather special interest as a universal and feasible add-on therapy for myocardial infarction (MI). In particular, human umbilical cord matrix-derived mesenchymal stromal cells (UCM-MSC) are advantageous since can be easily obtained and display high expansion potential. Using isolation protocols compliant with cell therapy, we previously showed UCM-MSC preserved cardiac function and attenuated remodeling 2 weeks after MI. In this study, UCM-MSC from two umbilical cords, UC-A and UC-B, were transplanted in a murine MI model to investigate consistency and durability of the therapeutic benefits. Both cellular products improved cardiac function and limited adverse cardiac remodeling 12 weeks post-ischemic injury, supporting sustained and long-term beneficial therapeutic effect. Donor associated variability was found in the modulation of cardiac remodeling and activation of the Akt-mTOR-GSK3ß survival pathway. In vitro, the two cell products displayed similar ability to induce the formation of vessel-like structures and comparable transcriptome in normoxia and hypoxia, apart from UCM-MSCs proliferation and expression differences in a small subset of genes associated with MHC Class I. These findings support that UCM-MSC are strong candidates to assist the treatment of MI whilst calling for the discussion on methodologies to characterize and select best performing UCM-MSC before clinical application.

Long-term simulation of a full-scale EBPR plant with a novel metabolic-ASM model and its use as a diagnostic tool.

This study evaluates the predictive capacity of the META-ASM model, a new integrated metabolic activated sludge model, in describing the long-term performance of a full-scale enhanced biological phosphorus removal (EBPR) system that suffers from inconsistent performance. In order to elucidate the causes of EBPR upsets and troubleshoot the process accordingly, the META-ASM model was tested as an operational diagnostic tool in a 1336-day long-term dynamic simulation, while its performance was compared with the ASM-inCTRL model, a version based on the Barker & Dold model. Overall, the predictions obtained with the META-ASM without changing default parameters were more reliable and effective at describing the active biomass of polyphosphate accumulating organisms (PAOs) and the dynamics of their storage polymers. The primary causes of the EBPR upsets were the high aerobic hydraulic retention times (HRTs) and low organic loading rates (OLRs) of the plant, which led to periods of starvation. The impact of these factors on EBPR performance were only identified with the META-ASM model. Furthermore, the first signs of process upsets were predicted by variations in the aerobic PAO maintenance rates, suggesting that the META-ASM model has potential to provide an early warning of process upset. The simulation of a new viable operational strategy indicated that troubleshooting the process could be achieved by reducing the aerated volume by switching off air in the first half of the aeration tank. In this new strategy, the META-ASM model predicted a simultaneous improvement in the biological phosphorus (P) and nitrogen (N) removal due to the enhancement of the hydrolysis and fermentation of the mixed liquor sludge in the new unaerated zone, which increased the availability of volatile fatty acids (VFAs) for PAOs. This study demonstrates that the META-ASM model is a powerful operational diagnostic tool for EBPR systems, capable of predicting and mitigating upsets, optimising performance and evaluating new process designs.

A novel metabolic-ASM model for full-scale biological nutrient removal systems.

This study demonstrates that META-ASM, a new integrated metabolic activated sludge model, provides an overall platform to describe the activity of the key organisms and processes relevant to biological nutrient removal (BNR) systems with a robust single-set of default parameters. This model overcomes various shortcomings of existing enhanced biological phosphorous removal (EBPR) models studied over the last twenty years. The model has been tested against 34 data sets from enriched lab polyphosphate accumulating organism (PAO)-glycogen accumulating organism (GAO) cultures and experiments with full-scale sludge from five water resource recovery facilities (WRRFs) with two different process configurations: three stage Phoredox (A2/O) and adapted Biodenitro™ combined with a return sludge sidestream hydrolysis tank (RSS). Special attention is given to the operational conditions affecting the competition between PAOs and GAOs, capability of PAOs and GAOs to denitrify, metabolic shifts as a function of storage polymer concentrations, as well as the role of these polymers in endogenous processes and fermentation. The overall good correlations obtained between the predicted versus measured EBPR profiles from different data sets support that this new model, which is based on in-depth understanding of EBPR, reduces calibration efforts. On the other hand, the performance comparison between META-ASM and literature models demonstrates that existing literature models require extensive parameter changes and have limited predictive power, especially in the prediction of long-term EBPR performance. The development of such a model able to describe in detail the microbial and chemical transformations of BNR systems with minimal adjustment to parameters suggests that the META-ASM model is a powerful tool to predict and mitigate EBPR upsets, optimise EBPR performance and to evaluate new process designs.

Is There an Ideal Rest Interval Between Races During Vaquejada in Which It Would Be Possible to Associate Best Performance and Welfare?

Vaquejada is an important Brazilian equine discipline. Understanding physiological adaptations of these athletes is crucial to improve properly performance, guaranteeing welfare. The objective of this study was to evaluate the influence of three vaquejada simulation tests (VST) on physiological parameters of horses and standardize a possible rest interval between races. Ten clinically healthy Quarter horses, 8.9 ± 4.3 year-old and 441.3 ± 25.0 kg, executed three VST, 5 days apart from each other. Vaquejada simulation tests consisted of two horses, a puller, and a helper, running with a bull on a soft sand track in which they must put the bull down. On M1, they ran three times with a 5-min rest between races; on M2, with a 10-min rest; and M3, with a 15-min rest. Clinical evaluation and blood sampling were made in all VST, before (T0), immediately after first run (T1), second run (T2), third run (T3) and at 30 minutes (T4), and 4 hours (T5) of recovery. Variables were statistically analyzed with a bifactorial comparison (P < .05). Exercise increased heart rate (HR), respiratory rate, body temperature (BT), lactate, triglycerides, packed cell volume, RBC, and hemoglobin concentration, with higher values in pull horses due to a more intense exercise. With 15-min of rest interval, helper horses showed lower values of glucose, aspartate aminotransferase, creatine kinase, BT, and higher values of triglycerides, also working at the same speed and distance with a lower HRmax and HRmed. Pull and helper horses had shown modifications of biomarkers. Furthermore, 15-min rest interval between races improved performance of helper horses as they used properly energy sources and cardiovascular function, respecting precepts of welfare.

Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in Plasmodium berghei but ITPase and NDH are dispensable.

BACKGROUND: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. METHODS: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). RESULTS: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. CONCLUSIONS: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.

The Secretome Derived From 3D-Cultured Umbilical Cord Tissue MSCs Counteracts Manifestations Typifying Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder whose treatment is mostly restricted to pain and symptom management and to the delay of joint destruction. Mesenchymal stem/stromal cells from the umbilical cord tissue (UC-MSCs) have previously been proven to be immunomodulatory and more efficient than bone marrow-derived MSCs in causing remission of local and systemic arthritic manifestations in vivo. Given the paracrine nature of UC-MSC activity, their application as active substances can be replaced by their secretome, thus avoiding allogeneic rejection and safety issues related to unwanted grafting. In this work, we aimed at demonstrating the viability of applying the 3D-primed UC-MSC secretome for the amelioration of arthritic signs. A proteomic analysis was performed to both, media conditioned by UC-MSC monolayer (CM2D) and 3D cultures (CM3D). The analysis of relevant trophic factors confirmed secretome profiles with very significant differences in terms of therapeutic potential. Whereas, CM3D was characterised by a prevailing expression of anti-inflammatory cytokines such as IL-10 and LIF, along with trophic factors involved in different mechanisms leading to tissue regeneration, such as PDGF-BB, FGF-2, I-309, SCF, and GM-CSF; CM2D presented relatively higher levels of IL-6, MCP-1, and IL-21, with recognised pro-inflammatory roles in joint disease and pleiotropic effects in the progression of rheumatoid arthritis (RA). Accordingly, different motogenic effects over mouse chondrocytes and distinct capacities of inducing glycosaminoglycan synthesis in vitro were observed between CM3D and CM2D. Finally, the evaluation of arthritic manifestations in vivo, using an adjuvant-induced model for arthritis (AIA), suggested a significantly higher therapeutic potential of CM3D over CM2D and even UC-MSCs. Histological analysis confirmed a faster remission of local and systemic arthritic manifestations of CM3D-treated animals. Overall, the results show that the use of UC-MSC CM3D is a viable and better strategy than direct UC-MSC administration for counteracting AIA-related signs. This strategy represents a novel MSC-based but nonetheless cell-free treatment for arthritic conditions such as those characterising RA.

LEGClust- a clustering algorithm based on layered entropic subgraphs.

Hierarchical clustering is a stepwise clustering method usually based on proximity measures between objects or sets of objects from a given data set. The most common proximity measures are distance measures. The derived proximity matrices can be used to build graphs, which provide the basic structure for some clustering methods. We present here a new proximity matrix based on an entropic measure and also a clustering algorithm (LEGClust) that builds layers of subgraphs based on this matrix, and uses them and a hierarchical agglomerative clustering technique to form the clusters. Our approach capitalizes on both a graph structure and a hierarchical construction. Moreover, by using entropy as a proximity measure we are able, with no assumption about the cluster shapes, to capture the local structure of the data, forcing the clustering method to reflect this structure. We present several experiments on artificial and real data sets that provide evidence on the superior performance of this new algorithm when compared with competing ones.

A Plasmodium berghei sporozoite-based vaccination platform against human malaria.

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.

Compatible solutes from hyperthermophiles improve the quality of DNA microarrays.

BACKGROUND: DNA microarrays are among the most widely used technical platforms for DNA and RNA studies, and issues related to microarrays sensitivity and specificity are therefore of general importance in life sciences. Compatible solutes are derived from hyperthermophilic microorganisms and allow such microorganisms to survive in environmental and stressful conditions. Compatible solutes show stabilization effects towards biological macromolecules, including DNA. RESULTS: We report here that compatible solutes from hyperthermophiles increased the performance of the hybridization buffer for Affymetrix GeneChip(R) arrays. The experimental setup included independent hybridizations with constant RNA over a wide range of compatible solute concentrations. The dependence of array quality and compatible solute was assessed using specialized statistical tools provided by both the proprietary Affymetrix quality control system and the open source Bioconductor suite. CONCLUSION: Low concentration (10 to 25 mM) of hydroxyectoine, potassium mannosylglycerate and potassium diglycerol phosphate in hybridization buffer positively affected hybridization parameters and enhanced microarrays outcome. This finding harbours a strong potential for the improvement of DNA microarray experiments.

Malaria parasite LIMP protein regulates sporozoite gliding motility and infectivity in mosquito and mammalian hosts.

Gliding motility allows malaria parasites to migrate and invade tissues and cells in different hosts. It requires parasite surface proteins to provide attachment to host cells and extracellular matrices. Here, we identify the Plasmodium protein LIMP (the name refers to a gliding phenotype in the sporozoite arising from epitope tagging of the endogenous protein) as a key regulator for adhesion during gliding motility in the rodent malaria model P. berghei. Transcribed in gametocytes, LIMP is translated in the ookinete from maternal mRNA, and later in the sporozoite. The absence of LIMP reduces initial mosquito infection by 50%, impedes salivary gland invasion 10-fold, and causes a complete absence of liver invasion as mutants fail to attach to host cells. GFP tagging of LIMP caused a limping defect during movement with reduced speed and transient curvature changes of the parasite. LIMP is an essential motility and invasion factor necessary for malaria transmission.