Lipids and bioactive lipid mediators are essential for platelet function. The lipid profile of platelets is highly dynamic due to free exchange of lipids with the plasma, release of extracellular ...vesicles, and both enzymatic and nonenzymatic lipid conversion. The lipidome of platelets changes in response to activation to accommodate the functional requirements of platelets, particularly for maintenance of hemostasis. Furthermore, when stored at room temperature as a component for transfusion, the lipid profile of platelets is altered. Although there is a growing interest in alternate storage conditions, such as refrigeration and cryopreservation, few contemporary studies have examined the impact of these storage modes on the lipid profile. However, evidence exists that bioactive lipid mediators produced over the storage of blood products may have functional implications once these products are transfused. As such, there is a need to determine the changes occurring to the lipid profile of these products over storage. This review outlines the role of lipids in platelets and discusses the current state of lipidomics for studying platelet components for transfusion in an effort to highlight the necessity for additional transfusion-focused investigations.
•Lipids are necessary for platelet function.•The lipidome of platelets is altered by room temperature storage.•Changes to the lipidome are associated with adverse transfusion reactions.•Alternative storage conditions may alter the lipidome of the platelet component.•Scope exists to research the lipidome of alternatively stored platelet components.
Background and Objectives
Refrigeration (cold‐storage) of pathogen inactivated (PI) platelet components may increase the shelf‐life and safety profile of platelet components, compared to conventional ...room‐temperature (RT) storage. Whilst there is substantial knowledge regarding the impact of these individual treatments on platelets, the combined effect has not been assessed.
Materials and methods
Using a pool‐and‐split study design, paired buffy‐coat derived platelets in 70% platelet additive solution (SSP+; MacoPharma) were left untreated or PI‐treated using the THERAFLEX UV‐Platelets System (UVC; MacoPharma). Units from each pair were split and stored at room temperature (20–24°C) or cold‐stored (2–6°C) to yield RT, cold, RT‐UVC and cold‐UVC study groups (n = 8 in each group). In vitro quality and function was tested over 9 days.
Results
Cold‐storage of UVC‐treated platelets reduced glycolytic metabolism (glucose consumption and lactate production) compared to RT‐UVC units. Cold‐UVC platelets demonstrated complete abrogation of HSR by day 5, increased externalisation of phosphatidylserine (annexin‐V binding) and activation of the GPIIb/IIIa receptor (PAC‐1 binding) above the levels observed with the individual treatments. Aggregation responses (ADP and collagen) were enhanced in the cold‐UVC platelets compared to both RT groups, but this was primarily mediated by cold‐storage. Haemostatic function, as measured using TEG, was similar between the groups.
Conclusion
Cold‐storage of UVC‐treated platelets reduced PI‐induced acceleration of glycolytic metabolism. However, combining cold‐storage and UVC‐treatment resulted in additional phenotypic changes compared to each treatment individually. Further work is required to understand the impact of these changes in clinical efficacy.
The green microalga
Chlamydomonas reinhardtii
is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell ...wall–deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of
C. reinhardtii
strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor,
C. reinhardtii
strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory.
Key points
•
Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii
•
Unassembled cell wall pherophorins are major components of extracellular protein aggregates
•
Known aggregate composition informs future strategies for recombinant protein purification
Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental ...aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for
- and
-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.
Rationale
Matrix‐assisted laser desorption ionisation with mass spectrometry imaging (MSI) has seen rapid development in recent years and as such is becoming an important technique for the mapping of ...biomolecules from the surface of tissues. One key area of development is the optimisation of analyte extraction by using modified matrices or mixes of common ones.
Methods
A series of serial sections were prepared for lipid MSI by either dry coating (sublimation) or by wet spray application of several matrices. These samples were then evaluated for analyte extraction, delocalisation and dynamic range.
Results
We have shown that the spraying and sublimation methods of matrix application can be used complementarily. This creates large datasets, with each preparation method applied narrowly and then interpreted as a ‘fraction’ of the whole. Once combined, the dynamic range is significantly increased. We have dubbed this technique ‘matrix phase fractionation’.
Conclusions
We have found that, by utilising matrix phase fractionation for the detection of lipids in brain tissue, it is possible to create a significantly more comprehensive dataset than would otherwise be possible with traditional ‘single‐run’ workflows.
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel ...electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform's abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism's proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.
Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, ...which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me2SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me2SO at −30 °C or with 3% Me2SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me2SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me2SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me2SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me2SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.
Background and Objectives
Cold‐stored platelets are currently under clinical evaluation and have been approved for limited clinical use in the United States. Most studies have focused on the ...haemostatic functionality of cold‐stored platelets; however, limited information is available examining changes to their immune function.
Materials and Methods
Two buffy‐coat‐derived platelet components were combined and split into two treatment arms: room temperature (RT)‐stored (20–24°C) or refrigerated (cold‐stored, 2–6°C). The concentration of select soluble factors was measured in the supernatant using commercial ELISA kits. The abundance of surface receptors associated with immunological function was assessed by flow cytometry. Platelet aggregation was assessed in response to Escherichia coli and Staphylococcus aureus, in the presence and absence of RGDS (blocks active conformation of integrin α2β3).
Results
Cold‐stored platelet components contained a lower supernatant concentration of C3a, RANTES and PF4. The abundance of surface‐bound P‐selectin and integrin α2β3 in the activated conformation increased during cold storage. In comparison, the abundance of CD86, CD44, ICAM‐2, CD40, TLR1, TLR2, TLR4, TLR3, TLR7 and TLR9 was lower on the surface membrane of cold‐stored platelets compared to RT‐stored components. Cold‐stored platelets exhibited an increased responsiveness to E. coli‐ and S. aureus‐induced aggregation compared to RT‐stored platelets. Inhibition of the active conformation of integrin α2β3 using RGDS reduced the potentiation of bacterial‐induced aggregation in cold‐stored platelets.
Conclusion
Our data highlight that cold storage changes the in vitro immune characteristics of platelets, including their sensitivity to bacterial‐induced aggregation. Changes in these immune characteristics may have clinical implications post transfusion.
Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential ...bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5–95% of peptides in the ranges 1–4 and 1–8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.
The accurate quantification of changes in the abundance of proteins is one of the main applications of proteomics. The maintenance of accuracy can be affected by bias and error that can occur at many ...points in the experimental process, and normalization strategies are crucial to attempt to overcome this bias and return the sample to its regular biological condition, or normal state. Much work has been published on performing normalization on data post-acquisition with many algorithms and statistical processes available. However, there are many other sources of bias that can occur during experimental design and sample handling that are currently unaddressed. This article aims to cast light on the potential sources of bias and where normalization could be applied to return the sample to its normal state. Throughout we suggest solutions where possible but, in some cases, solutions are not available. Thus, we see this article as a starting point for discussion of the definition of and the issues surrounding the concept of normalization as it applies to the proteomic analysis of biological samples. Specifically, we discuss a wide range of different normalization techniques that can occur at each stage of the sample preparation and analysis process.
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