Publications by authors named "Elena Levin"

47 Publications

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Mol Plant Pathol 2021 01 10;22(1):117-129. Epub 2020 Nov 10.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion, Israel.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.1111/mpp.13013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749749PMC
January 2021

Risk analysis of transfusion of cryoprecipitate without consideration of ABO group.

Transfusion 2021 Jan 10;61(1):29-34. Epub 2020 Oct 10.

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

Background: Transfusion medicine standards in Canada state that adult recipients can be transfused with cryoprecipitate of any ABO group, however, not all hospitals follow this guideline. There is a paucity of data on cryoprecipitate anti-A/B levels to reinforce standards.

Study Design And Methods: Manual tube antibody titration was performed on 7 units of group O plasma and the corresponding cryosupernatant plasma and cryoprecipitate. IgG/IgM levels were determined by nephelometry. Additionally, 10 cryoprecipitate each from groups A, B, and O were similarly assessed. From the antibody titer distribution among these samples, the probability of making a pool of cryoprecipitate with a titer ≥1:100 was calculated using bootstrap analysis.

Results: Anti-A/B titers in cryoprecipitate were equivalent to those in corresponding plasma; partitioning of anti-A/B activity into cryoprecipitate was not observed. Average IgM concentration was higher in cryoprecipitate than in plasma (P < .01). However, no correlation between IgM levels and anti-A/B titers was established. Among 30 cryoprecipitates from routine blood bank inventory, the median antibody titer and mode were 1:32 and 1:16, respectively. Of the samples tested, 4 of 30 and 9 of 30 had titers above 1:100 and 1:50, respectively. The probability of transfusing an adult dose of cryoprecipitate (pool of 10 cryoprecipitate) with a titer higher than 1:100 was calculated to be less than 1 in 3 million.

Conclusions: This study provides strong evidence to support current Canadian transfusion medicine standards on the safety of transfusion of cryoprecipitate without the need for blood group matching in adult recipients.
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http://dx.doi.org/10.1111/trf.16125DOI Listing
January 2021

Multiple transcriptomic analyses and characterization of pathogen-related core effectors and LysM family members reveal their differential roles in fungal growth and pathogenicity in Penicillium expansum.

Mol Genet Genomics 2020 Nov 12;295(6):1415-1429. Epub 2020 Jul 12.

School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China.

Penicillium expansum is a destructive phytopathogen causing postharvest decay on many stored fruits. To develop effective and safe management strategies, it is important to investigate its pathogenicity-related mechanisms. In this study, a bioinformatic pipeline was constructed and 50 core effector genes were identified in P. expansum using multiple RNA-seq data sets and their putative functions were implicated by comparatively homologous analyses using pathogen-host interaction database. To functionally characterize P. expansum LysM domain proteins during infection, null mutants for the 15 uncharacterized putative LysM effectors were constructed and the fungal growth rate on either PDA or Cazpek medium or lesion expansion rate on the infected apple fruits was evaluated. The results showed the growth rate of knockout mutants from PeLysM5, PeLysM12 and PeLysM15 was retarded on PDA medium. No significant difference in growth rate was observed between wild type and all mutants on solid Cazpek medium. Nevertheless, the hypha of wild type displayed deeper yellow on the back of Cazpek medium than those of knockout mutants. On the infecting apples fruits, the knockout mutants from PeLysM5, PeLysM7, PeLysM8, PeLysM9, PeLysM10, PeLysM11, PeLysM14, PeLysM15, PeLysM16, PeLysM18 and PeLysM19 showed enhanced fungal virulence, with faster decaying on infected fruits than those from wild type. By contrast, the knockout mutation at PeLysM12 locus led to reduced lesion expansion rate on the infected apple fruits. In addition, P. expansum-apple interaction RNA-seq experiment was performed using apple fruit tissues infected by the wild type and knockout mutant ΔPeLysM15, respectively. Transcriptome analyses indicated that deletion of PeLysM15 could activate expression of several core effector genes, such as PEX2_055830, PEX2_036960 and PEX2_108150, and a chitin-binding protein, PEX2_064520. These results suggest PeLysM15 may play pivotal roles in fungal growth and development and involve pathogen-host interaction by modulating other effector genes' expression. Our results could provide solid data reference and good candidates for further pathogen-related studies in P. expansum.
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http://dx.doi.org/10.1007/s00438-020-01710-9DOI Listing
November 2020

Improved in vitro quality of stored red blood cells upon oxygen reduction prior to riboflavin/UV light treatment of whole blood.

Transfusion 2019 10 13;59(10):3197-3204. Epub 2019 Aug 13.

Centre for Innovation, Canadian Blood Services, University of British Columbia, Vancouver, British Columbia, Canada.

Background: The application of riboflavin/UV-based pathogen inactivation (PI) to whole blood (WB) is currently limited by its negative impact on red blood cell (RBC) quality. The generation of reactive oxidative species in RBC products contributes to increased hemolysis. This study evaluated the impact of deoxygenation of WB prior to riboflavin/UV light treatment versus deoxygenation of RBC concentrates after PI treatment by monitoring RBC in vitro quality parameters.

Study Design And Methods: Six ABO-matched WB units were pooled and split. Within three pairs, one unit was treated with riboflavin/UV light while the other was kept as an untreated control prior to manufacture into red cell concentrates (RCCs). The first pair (Cntr; Cntr-PI) served as the normoxic controls. Deoxygenation was performed at the RCC level for the second pair (RCCdeox; PI-RCCdeox), and at the WB level of the third pair (WBdeox; WBdeox-PI). In vitro qualities of the respective RBC units were assessed throughout storage.

Results: The data for the Cntr and Cntr-PI units were comparable to previous reports. The PI-RCCdeox units exhibited worse in vitro quality for most parameters tested compared to Cntr-PI and WBdeox-PI units throughout storage. Hemolysis and microvesicle release was significantly (p < 0.05) higher on Days 21 and 42 in Cntr-PI units compared to WBdeox-PI units.

Conclusion: WB deoxygenation may help to decrease the accelerated deterioration in RCC in vitro quality caused by treatment with riboflavin/UV light. Treatment of WB under reduced oxygen levels needs to be assessed for PI effectiveness.
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http://dx.doi.org/10.1111/trf.15485DOI Listing
October 2019

Identification and Functional Analysis of NLP-Encoding Genes from the Postharvest Pathogen .

Microorganisms 2019 Jun 15;7(6). Epub 2019 Jun 15.

Department of Postharvest Science, Agricultural Research Organization, the Volcani Center, Rishon LeZion 7505101, Israel.

is a major postharvest pathogen that infects different fruits, mainly through injuries inflicted during harvest or subsequent handling after harvest. Several effectors were suggested to mediate pathogenicity of in fruit tissue. Among these effectors Nep1-like proteins (NLPs), produced by various microorganisms with different lifestyles, are known for their ability to induce necrosis in dicot plants and were shown to be involved in virulence of several plant-related pathogens. This study was aimed at the identification and functional characterization of two NLP genes found in the genome of . The genes were designated and and were found to code type1 and type3 NLP respectively. Necrosis-inducing activity of the two proteins was demonstrated by transient expression in leaves. While expression was induced during apple infection and in liquid culture, the highest level of expression was found in ungerminated spores. Deletion of , but not , resulted in reduced virulence on apples manifested by reduced rate of lesion development (disease severity).
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http://dx.doi.org/10.3390/microorganisms7060175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616513PMC
June 2019

Concealed ester formation and amino acid metabolism to volatile compounds in table grape (Vitis vinifera L.) berries.

Plant Sci 2018 Sep 24;274:223-230. Epub 2018 May 24.

Department of Vegetable Crops, Newe Ya'ar Research Center, Ramat Yishay 30095, Israel. Electronic address:

Volatile esters contribute to the aroma and flavor of many fruits but are normally absent in grape berries (Vitis vinifera L.). To examine the biosynthetic potential of grape berries to form volatile esters, berry sections were incubated with exogenous L-Phe, L-Leu or L-Met. In general, amino-acid incubation caused the accumulation of the respective aldehydes and alcohols. Moreover, L-Leu incubation resulted in the accumulation of 3-methylbutyl acetate and L-Phe incubation resulted in the accumulation 2-phenylethyl acetate in 'Muscat Hamburg' but not in the other grape accessions. Exogenous L-Met administration did not result in volatile esters accumulation but the accumulation of sulfur volatile compounds such as methional and dimethyl disulfide was prominent. Berry-derived cell-free extracts displayed differential alcohol acetyltransferase activities and supported the formation of 3-methylbutyl acetate and benzyl acetate. 2-Phenylethyl acetate was produced only in 'Muscat Hamburg' cell-free extracts. VvAAT2, a newly characterized gene, was preferentially expressed in 'Muscat Hamburg' berries and functionally expressed in E. coli. VvAAT2 possesses alcohol acetyltransferase activity utilizing benzyl alcohol, 2-phenylethanol, hexanol or 3-methylbutanol as substrates. Our study demonstrates that grape berries have a concealed potential to accumulate volatile esters and this process is limited by substrate availability.
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http://dx.doi.org/10.1016/j.plantsci.2018.05.020DOI Listing
September 2018

Genome Sequence, Assembly and Characterization of Two Strains Used as Biocontrol Agents of Postharvest Diseases.

Front Microbiol 2018 3;9:593. Epub 2018 Apr 3.

Department of Postharvest Science, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel.

The yeast was reported as an efficient biological control agent of postharvest diseases of fruits and vegetables, and it is the bases of the commercial formulated product "Shemer." Several mechanisms of action by which inhibits postharvest pathogens were suggested including iron-binding compounds, induction of defense signaling genes, production of fungal cell wall degrading enzymes and relatively high amounts of superoxide anions. We assembled the whole genome sequence of two strains of using PacBio and Illumina shotgun sequencing technologies. Using the PacBio, a high-quality draft genome consisting of 93 contigs, with an estimated genome size of approximately 26 Mb, was obtained. Comparative analysis of proteins with the other three available closely related genomes revealed a shared core of homologous proteins coded by 5,776 genes. Comparing the genomes of the two strains using a SNP calling approach resulted in the identification of 564,302 homologous SNPs with 2,004 predicted high impact mutations. The size of the genome is exceptionally high when compared with those of available closely related organisms, and the high rate of homology among genes points toward a recent whole-genome duplication event as the cause of this large genome. Based on the assembled genome, sequences were annotated with a gene description and gene ontology (GO term) and clustered in functional groups. Analysis of CAZymes family genes revealed 1,145 putative genes, and transcriptomic analysis of CAZyme expression levels in during its interaction with either grapefruit peel tissue or revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue.
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http://dx.doi.org/10.3389/fmicb.2018.00593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891927PMC
April 2018

Transcriptomic Response of Resistant (PI613981-) and Susceptible ("Royal Gala") Genotypes of Apple to Blue Mold () Infection.

Front Plant Sci 2017 16;8:1981. Epub 2017 Nov 16.

United States Department of Agriculture-Agricultural Research Service, Kearneysville, WV, United States.

from Central Asia is a progenitor of the modern domesticated apple ( × ). Several accessions of are highly resistant to the postharvest pathogen . A previous study identified the qM-.1 QTL on LG3 for resistance to in the mapping population GMAL4593, developed using the resistant accession, -PI613981, and the susceptible cultivar "Royal Gala" (RG) (), as parents. The goal of the present study was to characterize the transcriptomic response of susceptible RG and resistant PI613981 apple fruit to wounding and inoculation with using RNA-Seq. Transcriptomic analyses 0-48 h post inoculation suggest a higher basal level of resistance and a more rapid and intense defense response to wounding and wounding plus inoculation with in -PI613981 than in RG. Functional analysis showed that ethylene-related genes and genes involved in "jasmonate" and "MYB-domain transcription factor family" were over-represented in the resistant genotype. It is suggested that the more rapid response in the resistant genotype (PI613981) plays a major role in the resistance response. At least twenty DEGs were mapped to the qM-3.1 QTL ( × v.1: 26,848,396-28,424,055) on LG3, and represent potential candidate genes responsible for the observed resistance QTL in -PI613981. RT-qPCR of several of these genes was used to validate the RNA-Seq data and to confirm their higher expression in MS0.
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http://dx.doi.org/10.3389/fpls.2017.01981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696741PMC
November 2017

Identification and characterization of LysM effectors in Penicillium expansum.

PLoS One 2017 30;12(10):e0186023. Epub 2017 Oct 30.

Department of Postharvest Science, ARO, the Volcani Center, Bet Dagan, Israel.

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186023PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662087PMC
November 2017

Supernatant reduction of stored gamma-irradiated red blood cells minimizes potentially harmful substances present in transfusion aliquots for neonates.

Transfusion 2017 12 7;57(12):3009-3018. Epub 2017 Aug 7.

Department of Pathology and Laboratory Medicine and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.

Background: In neonate transfusion, the use of a dedicated red blood cell (RBC) unit decreases donor exposure. A separate safety measure involves gamma irradiation of the RBCs to abrogate the possibility of transfusion-associated graft-versus-host disease. However, in combination, storage of gamma-irradiated RBCs leads to accumulation of potentially harmful substances in the supernatant.

Study Design And Methods: For this study, RBCs were pooled and split into three study arms. Centrifugation or gravity was used to pack RBCs of matched units thereby reducing the amount of supernatant that would be present in neonate transfusion aliquots; these were compared to matched control units. Supernatant measurements of potassium, hemoglobin (Hb), RBC microvesicle (RMV) content, and mannitol were made in aliquots prepared weekly up to 21 days after gamma irradiation. RBC morphology and osmotic fragility were also assessed to determine if supernatant reduction methods affected the storage lesion.

Results: Potassium and mannitol were significantly decreased in transfusion aliquots prepared with either of the supernatant reduction methods. On Day 21, potassium levels from supernatant-reduced aliquots were below those of Day 7 control aliquots. A decrease in free Hb was only detected on Day 21 in centrifuged aliquots. RMVs were significantly reduced in centrifuged aliquots and significantly increased in gravity-settled aliquots. The only measurable effect on storage lesion was a small increase in osmotic fragility of the RBCs subjected to supernatant reduction.

Conclusion: Supernatant reduction by centrifugation effectively reduces potassium, mannitol, and RMVs in aliquots from gamma-irradiated RBCs stored up to 21 days.
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http://dx.doi.org/10.1111/trf.14270DOI Listing
December 2017

Altered timing of riboflavin and ultraviolet light pathogen inactivation improves platelet in vitro quality.

Transfusion 2017 08 12;57(8):2026-2034. Epub 2017 May 12.

Centre for Innovation, Canadian Blood Services, Vancouver, British Columbia, Canada.

Background: The platelet (PLT) storage lesion is in part caused by the collection and/or production process. Pathogen inactivation (PI) further accelerates its development leading to a reduced in vitro PLT functionality and hence quality. Although the treatment of PLT concentrates (PCs) with riboflavin and ultraviolet light PI should occur within 22 hours of collection, in this study the impact of treatment timing on in vitro PLT quality was investigated.

Study Design And Methods: Apheresis PCs were PI treated on the day of production or on Days 1, 3, or 4 of storage or left untreated as control. A panel of in vitro variables was used to monitor quality throughout 7-day storage, including metabolism, PLT activation, and release of microparticles. Changes in phosphorylation profiles of proteins in the lysate and levels of PLT factor 4, thrombospondin, and epidermal growth factor (EGF) in the releasate were analyzed by immunoblots or enzyme-linked immunosorbent assay.

Results: By Day 7 of storage, units illuminated on Day 4 showed a smaller impact of the PI process than units treated on the day of production or one day after on PLT quality such as PLT activation; metabolic activity; microvesicle and EGF release; and phosphorylation of p38, ERK, and HSP27. PCs treated on Day 3 of storage displayed an intermediate effect.

Conclusion: The timing of PI treatment of PCs influences in vitro PLT quality. Based on these results, timing recommendations should be reconsidered. If PI is applied, inventory management in blood banks might improve with a more flexible collection and treatment regime.
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http://dx.doi.org/10.1111/trf.14159DOI Listing
August 2017

Genotyping-by-sequencing markers facilitate the identification of quantitative trait loci controlling resistance to Penicillium expansum in Malus sieversii.

PLoS One 2017 3;12(3):e0172949. Epub 2017 Mar 3.

Department of Postharvest Science, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.

Blue mold caused by Penicillium expansum is the most important postharvest disease of apple worldwide and results in significant financial losses. There are no defined sources of resistance to blue mold in domesticated apple. However, resistance has been described in wild Malus sieversii accessions, including plant introduction (PI)613981. The objective of the present study was to identify the genetic loci controlling resistance to blue mold in this accession. We describe the first quantitative trait loci (QTL) reported in the Rosaceae tribe Maleae conditioning resistance to P. expansum on genetic linkage group 3 (qM-Pe3.1) and linkage group 10 (qM-Pe10.1). These loci were identified in a M.× domestica 'Royal Gala' X M. sieversii PI613981 family (GMAL4593) based on blue mold lesion diameter seven days post-inoculation in mature, wounded apple fruit inoculated with P. expansum. Phenotypic analyses were conducted in 169 progeny over a four year period. PI613981 was the source of the resistance allele for qM-Pe3.1, a QTL with a major effect on blue mold resistance, accounting for 27.5% of the experimental variability. The QTL mapped from 67.3 to 74 cM on linkage group 3 of the GMAL4593 genetic linkage map. qM-Pe10.1 mapped from 73.6 to 81.8 cM on linkage group 10. It had less of an effect on resistance, accounting for 14% of the experimental variation. 'Royal Gala' was the primary contributor to the resistance effect of this QTL. However, resistance-associated alleles in both parents appeared to contribute to the least square mean blue mold lesion diameter in an additive manner at qM-Pe10.1. A GMAL4593 genetic linkage map composed of simple sequence repeats and 'Golden Delicious' single nucleotide polymorphism markers was able to detect qM-Pe10.1, but failed to detect qM-Pe3.1. The subsequent addition of genotyping-by-sequencing markers to the linkage map provided better coverage of the PI613981 genome on linkage group 3 and facilitated discovery of qM-Pe3.1. A DNA test for qM-Pe3.1 has been developed and is currently being evaluated for its ability to predict blue mold resistance in progeny segregating for qM-Pe3.1. Due to the long juvenility of apple, the availability of a DNA test to screen for the presence of qM-Pe3.1 at the seedling stage will greatly improve efficiency of breeding apple for blue mold resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172949PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336245PMC
August 2017

Platelet concentrate functionality assessed by thromboelastography or rotational thromboelastometry.

Transfusion 2016 11 16;56(11):2790-2798. Epub 2016 Aug 16.

Department of Pathology and Laboratory Medicine and Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.

Background: There is poor correlation between in vivo platelet concentrate (PC) transfusion outcome and in vitro tests, which typically do not test the functional effectiveness of platelets (PLTs), but rather measure PLT characteristics. We hypothesize that the application of thromboelastography (TEG) or rotational thromboelastometry (ROTEM) to evaluate the procoagulant activity of stored PLTs can predict PLT quality and, ultimately, distinguish among hyper- and nonresponsive PCs. Additionally, we hypothesize that due to their procoagulant properties, PLT microvesicles (PMVs) contribute to the clot signature in these methods.

Study Design And Methods: After the TEG assays were validated, buffy coat PCs were evaluated during the storage time and reconstituted with frozen plasma to different PLT concentrations. Poor quality PCs were generated and assessed by TEG and other in vitro tests. The contribution of PMVs to the TEG clot signature was assessed.

Results: Hemostatic analysis showed no significant change in maximum amplitude (MA) during storage of PCs up to Day 10. On Day 8 of storage, PCs that had been manipulated to have poor quality showed a significant decrease in MA. PMV-rich samples contributed to a significant increase in MA, and PMV count showed a significant correlation with maximum clot formation (r = 0.51, p < 0.01).

Conclusion: TEG was optimized for use with buffy coat PCs, although it was found to lack sensitivity to detect normal storage-related quality changes. Hemostatic measurement was sufficiently sensitive to dissect PLT and PMV contributions to clot formation and to detect PCs stored under poor conditions.
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http://dx.doi.org/10.1111/trf.13760DOI Listing
November 2016

The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

Structure 2016 06 5;24(6):956-64. Epub 2016 May 5.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA. Electronic address:

The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters.
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http://dx.doi.org/10.1016/j.str.2016.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899283PMC
June 2016

X-ray structure of a mammalian stearoyl-CoA desaturase.

Nature 2015 Aug 22;524(7564):252-6. Epub 2015 Jun 22.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs. Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome b5, which regenerates the di-iron centre. To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the di-iron centre.
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http://dx.doi.org/10.1038/nature14549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689147PMC
August 2015

Whole blood treated with riboflavin and ultraviolet light: quality assessment of all blood components produced by the buffy coat method.

Transfusion 2015 Apr 29;55(4):815-23. Epub 2014 Oct 29.

Centre for Innovation, Canadian Blood Services, Vancouver, British Columbia, Canada.

Background: Pathogen inactivation (PI) technologies are currently licensed for use with platelet (PLT) and plasma components. Treatment of whole blood (WB) would be of benefit to the blood banking community by saving time and costs compared to individual component treatment. However, no paired, pool-and-split study directly assessing the impact of WB PI on the subsequently produced components has yet been reported.

Study Design And Methods: In a "pool-and-split" study, WB either was treated with riboflavin and ultraviolet (UV) light or was kept untreated as control. The buffy coat (BC) method produced plasma, PLT, and red blood cell (RBC) components. PLT units arising from the untreated WB study arm were treated with riboflavin and UV light on day of production and compared to PLT concentrates (PCs) produced from the treated WB units. A panel of common in vitro variables for the three types of components was used to monitor quality throughout their respective storage periods.

Results: PCs derived from the WB PI treatment were of significantly better quality than treated PLT components for most variables. RBCs produced from the WB treatment deteriorated earlier during storage than untreated units. Plasma components showed a 3% to 44% loss in activity for several clotting factors.

Conclusion: Treatment of WB with riboflavin and UV before production of components by the BC method shows a negative impact on all three blood components. PLT units produced from PI-treated WB exhibited less damage compared to PLT component treatment.
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http://dx.doi.org/10.1111/trf.12895DOI Listing
April 2015

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity.

Mol Plant Microbe Interact 2015 Mar;28(3):232-48

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.
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http://dx.doi.org/10.1094/MPMI-09-14-0261-FIDOI Listing
March 2015

Structure of urea transporters.

Subcell Biochem 2014 ;73:65-78

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.

Members of the urea transporter (UT) family mediate rapid, selective transport of urea down its concentration gradient. To date, crystal structures of two evolutionarily distant UTs have been solved. These structures reveal a common UT fold involving two structurally homologous domains that encircle a continuous membrane-spanning pore and indicate that UTs transport urea via a channel-like mechanism. Examination of the conserved architecture of the pore, combined with crystal structures of ligand-bound proteins, molecular dynamics simulations, and functional data on permeation and inhibition by a broad range of urea analogs and other small molecules, provides insight into the structural basis of urea permeation and selectivity.
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http://dx.doi.org/10.1007/978-94-017-9343-8_5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422644PMC
April 2015

Structure of a membrane-embedded prenyltransferase homologous to UBIAD1.

PLoS Biol 2014 Jul 22;12(7):e1001911. Epub 2014 Jul 22.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

Membrane-embedded prenyltransferases from the UbiA family catalyze the Mg2+-dependent transfer of a hydrophobic polyprenyl chain onto a variety of acceptor molecules and are involved in the synthesis of molecules that mediate electron transport, including Vitamin K and Coenzyme Q. In humans, missense mutations to the protein UbiA prenyltransferase domain-containing 1 (UBIAD1) are responsible for Schnyder crystalline corneal dystrophy, which is a genetic disease that causes blindness. Mechanistic understanding of this family of enzymes has been hampered by a lack of three-dimensional structures. We have solved structures of a UBIAD1 homolog from Archaeoglobus fulgidus, AfUbiA, in an unliganded form and bound to Mg2+ and two different isoprenyl diphosphates. Functional assays on MenA, a UbiA family member from E. coli, verified the importance of residues involved in Mg2+ and substrate binding. The structural and functional studies led us to propose a mechanism for the prenyl transfer reaction. Disease-causing mutations in UBIAD1 are clustered around the active site in AfUbiA, suggesting the mechanism of catalysis is conserved between the two homologs.
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http://dx.doi.org/10.1371/journal.pbio.1001911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106721PMC
July 2014

Recent progress on the structure and function of the TrkH/KtrB ion channel.

Curr Opin Struct Biol 2014 Aug 8;27:95-101. Epub 2014 Jul 8.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Members of the Superfamily of K(+) Transporters (SKT) are integral membrane proteins that mediate the uptake of ions into non-animal cells. Although these proteins are homologous to the well-characterized K(+) channel family, relatively little was known about their transport and gating mechanisms until the recent determination of crystal structures for two SKT proteins, TrkH and KtrB. These structures reveal that the SKT proteins are channels, containing a flexible loop in the middle of the permeation pathway that may act as a gate. Two different conformational changes have been observed for the associated gating rings, suggesting different mechanisms of regulation by the binding of nucleotides.
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http://dx.doi.org/10.1016/j.sbi.2014.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182332PMC
August 2014

Structural insight into the PTS sugar transporter EIIC.

Biochim Biophys Acta 2015 Mar 20;1850(3):577-85. Epub 2014 Mar 20.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Background: The enzyme IIC (EIIC) component of the phosphotransferase system (PTS) is responsible for selectively transporting sugar molecules across the inner bacterial membrane. This is accomplished in parallel with phosphorylation of the sugar, which prevents efflux of the sugar back across the membrane. This process is a key part of an extensive signaling network that allows bacteria to efficiently utilize preferred carbohydrate sources.

Scope Of Review: The goal of this review is to examine the current understanding of the structural features of the EIIC and how it mediates concentrative, selective sugar transport. The crystal structure of an N,N'-diacetylchitobiose transporter is used as a structural template for the glucose superfamily of PTS transporters.

Major Conclusions: Comparison of protein sequences in context with the known EIIC structure suggests that members of the glucose superfamily of PTS transporters may exhibit variations in topology. Despite these differences, a conserved histidine and glutamate appear to have roles shared across the superfamily in sugar binding and phosphorylation. In the proposed transport model, a rigid body motion between two structural domains and movement of an intracellular loop provide the substrate binding site with alternating access, and reveal a surface required for interaction with the phosphotransfer protein responsible for catalysis.

General Significance: The structural and functional data discussed here give a preliminary understanding of how transport in EIIC is achieved. However, given the great sequence diversity between varying glucose-superfamily PTS transporters and lack of data on conformational changes needed for transport, additional structures of other members and conformations are still required. This article is part of a Special Issue entitled: Structural biochemistry and biophysics of membrane proteins.
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http://dx.doi.org/10.1016/j.bbagen.2014.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169766PMC
March 2015

Structural basis of the alternating-access mechanism in a bile acid transporter.

Nature 2014 Jan 8;505(7484):569-73. Epub 2013 Dec 8.

1] Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA [2] Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA [3] Ion Channel Research and Drug Development Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China.

Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na(+)-dependent bile acid transporters involved in enterohepatic recirculation, the Na(+)-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBT(NM)) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na(+) and a taurocholic acid. However, the structural changes that bring bile acid and Na(+) across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na(+) and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved 'crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for Na(+).
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http://dx.doi.org/10.1038/nature12811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142352PMC
January 2014

Process improvement by eliminating mixing of whole blood units after an overnight hold prior to component production using the buffy coat method.

J Blood Transfus 2013 5;2013:154838. Epub 2013 Jun 5.

Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, ON, K1G 4J5, Canada K1G 4J5.

The elimination of a thorough manual mixing of whole blood (WB) which takes place following the overnight hold, but before the first centrifugation step, during buffy coat component production at Canadian Blood Services (CBS) was investigated. WB was pooled after donation and split. Pairs of platelet, red blood cell (RBC), and plasma components were produced, with half using the standard method and half using a method in which the mixing step was eliminated. Quality assessments included yield, pH, CD62P expression and morphology for platelets, hemoglobin, hematocrit, hemolysis, and supernatant K(+) for RBCs, and volume and factor VIII activity levels for plasma. All components, produced using either method, met CBS quality control criteria. There were no significant differences in platelet yield between components produced with and without mixing. A significant difference was seen for RBC hemolysis at expiry (P = 0.03), but for both groups, levels met quality control requirements. Noninferiority of components produced without mixing was confirmed for all parameters. Manual mixing is laborious and has a risk of repetitive strain for production staff and its significance is unclear. Elimination of this step will improve process efficiencies without compromising quality.
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http://dx.doi.org/10.1155/2013/154838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771126PMC
September 2013

Gating of the TrkH ion channel by its associated RCK protein TrkA.

Nature 2013 Apr;496(7445):317-22

Department of Physiology & Cellular Biophysics, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032, USA.

TrkH belongs to a superfamily of K(+) transport proteins required for growth of bacteria in low external K(+) concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K(+) channel and may have a gating mechanism substantially different from K(+) channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K(+)) domains, which are found in certain K(+) channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA.
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http://dx.doi.org/10.1038/nature12056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726529PMC
April 2013

Correction to Potentiation of the Kv1 family K(+) Channel by Cortisone Analogues.

ACS Chem Biol 2013 01;8(1):268

Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons and ‡Department of Psychiatry and Center for Molecular Recognition, Columbia University , 630 West 168th Street, New York, New York 10032, United States.

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http://dx.doi.org/10.1021/cb300701eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573369PMC
January 2013

Kinetic bromine isotope effect: example from the microbial debromination of brominated phenols.

Anal Bioanal Chem 2013 Mar 7;405(9):2923-9. Epub 2012 Oct 7.

Zuckerberg Institute for Water Research, Department of Environmental Hydrology and Microbiology, Ben-Gurion University of the Negev, Negev, Israel.

The increasing use of kinetic isotope effects for environmental studies has motivated the development of new compound-specific isotope analysis techniques for emerging pollutants. Recently, high-precision bromine isotope analysis in individual brominated organic compounds was proposed, by the coupling of gas chromatography to a multi-collector inductively coupled plasma mass spectrometer using strontium as an external spike for instrumental bias correction. The present study, for the first time, demonstrates an application of this technique for determining bromine kinetic isotope effects during biological reaction, focusing on the reductive debromination of brominated phenols under anaerobic conditions. Results show bromine isotope enrichment factors (ε) of -0.76 ± 0.08, -0.46 ± 0.19, and -0.20 ± 0.06 ‰ for the debromination of 4-bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol, respectively. These values are rather low, yet still high enough to be obtained with satisfying certainty. This further implies that the analytical method may be also appropriate for future environmental applications.
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http://dx.doi.org/10.1007/s00216-012-6446-0DOI Listing
March 2013

Potentiation of the Kv1 family K(+) channel by cortisone analogues.

ACS Chem Biol 2012 10 23;7(10):1641-6. Epub 2012 Jul 23.

Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032, United States.

The Kv1 family voltage-dependent K(+) channels are essential for termination of action potentials in neurons and myocytes. These channels form a stable complex with their beta subunits (Kvβ), some of which inhibit channel activity. Cortisone potentiates Kv1 channel by binding to Kvβ and promoting its dissociation from the channel, but its half-maximum effective concentration is ∼46 μM. To identify corticosteroids that are more efficient than cortisone, we examined 25 cortisone analogues and found that fluticasone propionate potentiates channel current with a half-maximum effective concentration (EC(50)) of 37 ± 1.1 nM. Further studies showed that fluticasone propionate potentiates channel current by inducing dissociation of Kvβ, and docking of fluticasone propionate into the cortisone binding site reveals potential interactions that enhance the EC(50) value. Thus, fluticasone propionate provides a starting point for rational design of more efficient small-molecule compounds that increase Kv1 activity and affect the integrity of the Kv1-Kvβ complex.
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http://dx.doi.org/10.1021/cb300233yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567311PMC
October 2012

Structure and permeation mechanism of a mammalian urea transporter.

Proc Natl Acad Sci U S A 2012 Jul 25;109(28):11194-9. Epub 2012 Jun 25.

Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA.

As an adaptation to infrequent access to water, terrestrial mammals produce urine that is hyperosmotic to plasma. To prevent osmotic diuresis by the large quantity of urea generated by protein catabolism, the kidney epithelia contain facilitative urea transporters (UTs) that allow rapid equilibration between the urinary space and the hyperosmotic interstitium. Here we report the first X-ray crystal structure of a mammalian UT, UT-B, at a resolution of 2.36 Å. UT-B is a homotrimer and each protomer contains a urea conduction pore with a narrow selectivity filter. Structural analyses and molecular dynamics simulations showed that the selectivity filter has two urea binding sites separated by an approximately 5.0 kcal/mol energy barrier. Functional studies showed that the rate of urea conduction in UT-B is increased by hypoosmotic stress, and that the site of osmoregulation coincides with the location of the energy barrier.
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http://dx.doi.org/10.1073/pnas.1207362109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396522PMC
July 2012

In vitro platelet quality in storage containers used for pediatric transfusions.

Transfusion 2012 Aug 18;52(8):1703-14. Epub 2012 Jan 18.

Canadian Blood Services Research and Development, Edmonton, Alberta, Canada.

Background: The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach.

Study Design And Methods: Small volumes (50 mL) of single apheresis PLT components (APCs), collected on either CaridianBCT Trima or Haemonetics MCS+ instruments, were aliquoted on Days 2, 3, 4, and 5 postcollection into Fenwal PL1240 or 4R2014 bags or 60-mL polypropylene syringes. Samples were tested for in vitro quality at their recommended expiry times (4 hr for 4R2014 bags and syringes or Day 5 for PL1240 bags). Assays included pH, CD62P expression, and metabolic measures.

Results: CD62P expression increased throughout storage in all containers. Among the small-volume containers, pH, pCO(2) , lactate, and bicarbonate varied considerably. Regardless of the day of aliquoting, pCO(2) was significantly higher and pO(2) was significantly lower in gas-impermeable syringes than other containers. No bacterial growth was detected in any sample.

Conclusion: The quality of APCs aliquoted into small-volume containers meets regulatory requirements and is generally equivalent to that of full-volume APCs at expiry.
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http://dx.doi.org/10.1111/j.1537-2995.2011.03516.xDOI Listing
August 2012

Development of a quality monitoring program for platelet components: a report of the first four years' experience at Canadian Blood Services.

Transfusion 2012 Apr 7;52(4):810-8. Epub 2011 Nov 7.

Canadian Blood Services, UBC Centre for Blood Research, Vancouver, British Columbia, Canada.

Background: A quality monitoring program (QMP) for platelet concentrates (PCs) was implemented at Canadian Blood Services (CBS) to improve standards and to better understand platelet (PLT) products by supplementing routine quality control (QC).

Study Design And Methods: Annual surveys of PCs from CBS production sites were conducted, with four completed to date (QMP Cycles 1-4) spanning two different PC production methods: PLT-rich plasma (PRP) and buffy coat (BC). Randomly selected PCs were sent to a central laboratory and tested 1 day after expiry. An expanded panel of tests including CD62P expression by flow cytometry, mean PLT volume, PLT count and morphology, extent of shape change, and PLT metabolic parameters, were applied.

Results: QMP data on the implementation of the BC production method across CBS indicated that BC PCs have less variable in vitro quality measures than PRP PCs. For the QC parameters pH and PLT count per unit, the range of mean values from each site for QMP 3 and 4 fell well within the range defined by regulatory standards, a first step in defining quality benchmarks for PCs. Of the extended panel of quality parameters, CD62P expression was the most sensitive indicator of change and identified an issue with the implementation of the BC PC production method at one site, which was subsequently remedied.

Conclusion: A QMP was found to be useful to monitor production processes across sites and highlights best practice approaches while deepening understanding of the quality of PLT products at CBS.
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http://dx.doi.org/10.1111/j.1537-2995.2011.03402.xDOI Listing
April 2012