Structures of synaptic vesicle protein 2A and 2B bound to anticonvulsants.

Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s.

Physiological genetic variation in tomato fruit chilling tolerance during postharvest storage.

Storage at low temperatures is a common practice to prolong postharvest life of fruit and vegetables with a minimal negative impact on human/environmental health. Storage at low temperatures, however, can be restricted due to produce susceptibility to non-freezing chilling temperatures, when injuries such as physiological disorders and decays may result in unmarketable produce. We have investigated tomato fruit response to postharvest chilling stress in a recombinant inbred line (RIL) population developed from a cross between a chilling-sensitive cultivated tomato (Solanum lycopersicum L.) breeding line and a chilling-tolerant inbred accession of the tomato wild species S. pimpinellifolium L. Screening of the fruit of 148 RILs under cold storage (1.5°C) indicated presence of significant variations in chilling tolerance, manifested by varying degrees of fruit injury. Two extremely contrasting groups of RILs were identified, chilling-tolerant and chilling-sensitive RILs. The RILs in the two groups were further investigated under chilling stress conditions, and several physiological parameters, including weight loss, chlorophyll fluorescence parameters Fv/Fm, and Performance Index (PI), were determined to be efficient markers for identifying response to chilling stress in postharvest fruit. The Fv/Fm values reflected the physiological damages endured by the fruit after cold storage, and PI was a sensitive marker for early changes in photosystem II function. These two parameters were early indicators of chilling response before occurrence of visible chilling injuries. Antioxidant activities and ascorbic acid content were significantly higher in the chilling-tolerant than the chilling-sensitive lines. Further, the expression of C-repeat/DREB binding factors (CBFs) genes swiftly changed within 1-hr of fruit exposure to the chilling temperature, and the SlCBF1 transcript level was generally higher in the chilling-tolerant than chilling-sensitive lines after 2-hr exposure to the low temperature. This research demonstrates the presence of potential genetic variation in fruit chilling tolerance in the tomato RIL population. Further investigation of the RIL population is underway to better understand the genetic, physiological, and biochemical mechanisms involved in postharvest fruit chilling tolerance in tomato.

Effect of modern infusion pumps on RBC quality.

BACKGROUND: Mechanical stress on red blood cells is associated with using infusion pumps for blood administration. Current standards in North America leave it to healthcare facilities to consult with manufacturers about infusion pump safety for transfusion; studies on various pumps and red blood cell (RBC) conditions are scarce. STUDY DESIGN AND METHODS: RBC units were pumped through four infusion pumps on d22 (22 days postcollection), d40, d28 after gamma irradiation on d14 (I14d28), and d22 after irradiation on d21 (I21d22). For each experiment, three units were pooled and split among four bags. Samples were collected at gravity and after pumping at clinical nonemergency rates. Hemolysis %, microvesicles, potassium, lactate dehydrogenase, mechanical fragility index levels, and morphology evaluations were performed (n = 5-6). RESULTS: Hemolysis levels of Piston and Linear Peristaltic pump samples were not different from hemolysis of corresponding gravity samples. Peristaltic samples had significantly higher hemolysis compared to gravity, and other pumps, however, maximum mean difference was limited to 0.05%. Pumping at 50 mL/h resulted in the highest hemolysis level. Change in hemolysis % due to pumping was significantly higher in d40 and I21d22 units. No combination of pumps and RBCs conditions led to hemolysis >0.8%. Besides hemolysis, lactate dehydrogenase release was the only marker that demonstrated some differences between infusions via pump versus gravity. CONCLUSION: The pump design affects the degree of hemolysis. However, for all tested pumps and RBC conditions, this increase was minimal. Hemolysis measurement on d40 and I21d22 at 50 mL/h were concluded to be appropriate parameters for pump evaluation.

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

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.

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

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.

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.

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.

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

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.

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

Penicillium expansum 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 P. expansum 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 P. expansum. The genes were designated Penlp1 and Penlp2 and were found to code type1 and type3 NLP respectively. Necrosis-inducing activity of the two proteins was demonstrated by transient expression in Nicotiana benthamiana leaves. While Penlp1 expression was induced during apple infection and in liquid culture, the highest level of Penlp2 expression was found in ungerminated spores. Deletion of Penlp1, but not Penlp2, resulted in reduced virulence on apples manifested by reduced rate of lesion development (disease severity).

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

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.

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

The yeast Metschnikowia fructicola 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 M. fructicola 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 M. fructicola 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 M. fructicola 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 M. fructicola 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 M. fructicola 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 M. fructicola during its interaction with either grapefruit peel tissue or Penicillium digitatum revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue.

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

Malus sieversii from Central Asia is a progenitor of the modern domesticated apple (Malus × domestica). Several accessions of M. sieversii are highly resistant to the postharvest pathogen Penicillium expansum. A previous study identified the qM-Pe3.1 QTL on LG3 for resistance to P. expansum in the mapping population GMAL4593, developed using the resistant accession, M. sieversii -PI613981, and the susceptible cultivar "Royal Gala" (RG) (M. domestica), 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 P. expansum 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 P. expansum in M. sieversii -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 (Malus sieversii-PI613981) plays a major role in the resistance response. At least twenty DEGs were mapped to the qM-Pe3.1 QTL (M × d v.1: 26,848,396-28,424,055) on LG3, and represent potential candidate genes responsible for the observed resistance QTL in M. sieversii-PI613981. RT-qPCR of several of these genes was used to validate the RNA-Seq data and to confirm their higher expression in MS0.

Identification and characterization of LysM effectors in Penicillium expansum.

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.

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

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.

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

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.

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

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.

Platelet concentrate functionality assessed by thromboelastography or rotational thromboelastometry.

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.

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

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.

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

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.

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

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.

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

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.