Current Insights Into K-associated Fetal Anemia and Potential Treatment Strategies for Sensitized Pregnancies.

K-associated anemic disease of the fetus and newborn (K-ADFN) is a rare but life-threatening disease in which maternal alloantibodies cross the placenta and can mediate an immune attack on fetal red blood cells expressing the K antigen. A considerably more common disease, D-associated hemolytic disease of the fetus and newborn (D-HDFN), can be prophylactically treated using polyclonal α-D antibody preparations. Currently, no such prophylactic treatment exists for K-associated fetal anemia, and disease is usually treated with intrauterine blood transfusions. Here we review current understanding of the biology of K-associated fetal anemia, how the maternal immune system is sensitized to fetal red blood cells, and what is understood about potential mechanisms of prophylactic HDFN interventions. Given the apparent challenges associated with preventing alloimmunization, we highlight novel strategies for treating sensitized mothers to prevent fetal anemia that may hold promise not only for K-mediated disease, but also for other pathogenic alloantibody responses.

Impact of adjuvants on the biophysical and functional characteristics of HIV vaccine-elicited antibodies in humans.

Adjuvants can alter the magnitude, characteristics, and persistence of the humoral response to protein vaccination. HIV vaccination might benefit from tailored adjuvant choice as raising a durable and protective response to vaccination has been exceptionally challenging. Analysis of trials of partially effective HIV vaccines have identified features of the immune response that correlate with decreased risk, including high titers of V1V2-binding IgG and IgG3 responses with low titers of V1V2-binding IgA responses and enhanced Fc effector functions, notably antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, there has been limited opportunity to compare the effect of different adjuvants on these activities in humans. Here, samples from the AVEG015 study, a phase 1 trial in which participants (n = 112) were immunized with gp120SF-2 and one of six different adjuvants or combinations thereof were assessed for antibody titer, biophysical features, and diverse effector functions. Three adjuvants, MF59 + MTP-PE, SAF/2, and SAF/2 + MDP, increased the peak magnitude and durability of antigen-specific IgG3, IgA, FcγR-binding responses and ADCP activity, as compared to alum. While multiple adjuvants increased the titer of IgG, IgG3, and IgA responses, none consistently altered the balance of IgG to IgA or IgG3 to IgA. Linear regression analysis identified biophysical features including gp120-specific IgG and FcγR-binding responses that could predict functional activity, and network analysis identified coordinated aspects of the humoral response. These analyses reveal the ability of adjuvants to drive the character and function of the humoral response despite limitations of small sample size and immune variability in this human clinical trial.

Recent insights into Fc-mediated effector responses to HIV-1.

PURPOSE OF REVIEW: Recent work defining Fc-mediated effector functions for both viral control and protection against infection is summarized and considered along with new strategies to drive robust Fc-mediated responses. RECENT FINDINGS: In new human and nonhuman primate (NHP) vaccine trials as well as studies of natural infection, Fc-mediated effector responses have sometimes been observed to correlate with decreased risk of infection or with better clinical outcomes, suggesting a potential role for these responses in HIV-1 prevention and therapy. Recent highlights include use of antibody-dependent cellular cytotoxicity-sensitizing CD4-induced mimetic compounds, novel V1V2 immunogens, passive transfer studies, and vaccine regimens that successfully elicited Fc-mediated responses and were reported to decrease risk of infection in challenge studies in NHPs. Lastly, detailed studies of IgG3 forms of HIV-specific antibodies have reported that both neutralizing and Fc-mediated responses can be increased relative to the more prevalent IgG1 subclass. SUMMARY: Successful harmonization of neutralizing and Fc-mediated responses may make key contributions to the goal of reducing HIV-1 infection via active and passive vaccination. New studies continue to highlight the importance of Fc-mediated antibody responses as correlates of decreased risk of infection and suggest enhanced phagocytosis is a potential mechanism of reduced risk of infection associated with human IgG3 responses. Results from recent studies may help guide the rational design of therapies and vaccines that aim to specifically leverage antibody effector function.

Co-immunization of DNA and Protein in the Same Anatomical Sites Induces Superior Protective Immune Responses against SHIV Challenge.

We compare immunogenicity and protective efficacy of an HIV vaccine comprised of env and gag DNA and Env (Envelope) proteins by co-administration of the vaccine components in the same muscles or by separate administration of DNA + protein in contralateral sites in female rhesus macaques. The 6-valent vaccine includes gp145 Env DNAs, representing six sequentially isolated Envs from the HIV-infected individual CH505, and matching GLA-SE-adjuvanted gp120 Env proteins. Interestingly, only macaques in the co-administration vaccine group are protected against SHIV CH505 acquisition after repeated low-dose intravaginal challenge and show 67% risk reduction per exposure. Macaques in the co-administration group develop higher Env-specific humoral and cellular immune responses. Non-neutralizing Env antibodies, ADCC, and antibodies binding to FcγRIIIa are associated with decreased transmission risk. These data suggest that simultaneous recognition, processing, and presentation of DNA + Env protein in the same draining lymph nodes play a critical role in the development of protective immunity.

Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons.

Zinc (Zn2+) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn2+ plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn2+ sensors to rigorously define resting Zn2+ levels and stimulation-dependent intracellular Zn2+ dynamics, and we performed RNA-Seq to characterize Zn2+-dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn2+ during stimulation altered expression levels of 931 genes, and these Zn2+ dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn2+ in these cultures, we did detect the synaptic vesicle Zn2+ transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn2+ increases. These results provide the first global sequencing-based examination of Zn2+-dependent changes in transcription and identify genes that may mediate Zn2+-dependent processes and functions.

Genomic Selection for Ascochyta Blight Resistance in Pea.

Genomic selection (GS) is a breeding tool, which is rapidly gaining popularity for plant breeding, particularly for traits that are difficult to measure. One such trait is ascochyta blight resistance in pea (Pisum sativum L.), which is difficult to assay because it is strongly influenced by the environment and depends on the natural occurrence of multiple pathogens. Here we report a study of the efficacy of GS for predicting ascochyta blight resistance in pea, as represented by ascochyta blight disease score (ASC), and using nucleotide polymorphism data acquired through genotyping-by-sequencing. The effects on prediction accuracy of different GS models and different thresholds for missing genotypic data (which modified the number of single nucleotide polymorphisms used in the analysis) were compared using cross-validation. Additionally, the inclusion of marker × environment interactions in a genomic best linear unbiased prediction (GBLUP) model was evaluated. Finally, different ways of combining trait data from two field trials using bivariate, spatial, and single-stage analyses were compared to results obtained using a mean value. The best prediction accuracy achieved for ASC was 0.56, obtained using GBLUP analysis with a mean value for ASC and data quality threshold of 70% (i.e., missing SNP data in <30% of lines). GBLUP and Bayesian Reproducing kernel Hilbert spaces regression (RKHS) performed slightly better than the other models trialed, whereas different missing data thresholds made minimal differences to prediction accuracy. The prediction accuracies of individual, randomly selected, testing/training partitions were highly variable, highlighting the effect that the choice of training population has on prediction accuracy. The inclusion of marker × environment interactions did not increase the prediction accuracy for lines which had not been phenotyped, but did improve the results of prediction across environments. GS is potentially useful for pea breeding programs pursuing ascochyta blight resistance, both for predicting breeding values for lines that have not been phenotyped, and for providing enhanced estimated breeding values for lines for which trait data is available.

Association mapping of starch chain length distribution and amylose content in pea (Pisum sativum L.) using carbohydrate metabolism candidate genes.

BACKGROUND: Although starch consists of large macromolecules composed of glucose units linked by α-1,4-glycosidic linkages with α-1,6-glycosidic branchpoints, variation in starch structural and functional properties is found both within and between species. Interest in starch genetics is based on the importance of starch in food and industrial processes, with the potential of genetics to provide novel starches. The starch metabolic pathway is complex but has been characterized in diverse plant species, including pea. RESULTS: To understand how allelic variation in the pea starch metabolic pathway affects starch structure and percent amylose, partial sequences of 25 candidate genes were characterized for polymorphisms using a panel of 92 diverse pea lines. Variation in the percent amylose composition of extracted seed starch and (amylopectin) chain length distribution, one measure of starch structure, were characterized for these lines. Association mapping was undertaken to identify polymorphisms associated with the variation in starch chain length distribution and percent amylose, using a mixed linear model that incorporated population structure and kinship. Associations were found for polymorphisms in seven candidate genes plus Mendel's r locus (which conditions the round versus wrinkled seed phenotype). The genes with associated polymorphisms are involved in the substrate supply, chain elongation and branching stages of the pea carbohydrate and starch metabolic pathways. CONCLUSIONS: The association of polymorphisms in carbohydrate and starch metabolic genes with variation in amylopectin chain length distribution and percent amylose may help to guide manipulation of pea seed starch structural and functional properties through plant breeding.

Critical Comparison of FRET-Sensor Functionality in the Cytosol and Endoplasmic Reticulum and Implications for Quantification of Ions.

Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for quantifying and visualizing analytes in living cells, and when targeted to organelles have the potential to define distribution of analytes in different parts of the cell. However, quantitative estimates of analyte distribution require rigorous and systematic analysis of sensor functionality in different locations. In this work, we establish methods to critically evaluate sensor performance in different organelles and carry out a side-by-side comparison of three different genetically encoded sensor platforms for quantifying cellular zinc ions (Zn2+). Calibration conditions are optimized for high dynamic range and stable FRET signals. Using a combination of single-cell microscopy and a novel microfluidic platform capable of screening thousands of cells in a few hours, we observe differential performance of these sensors in the cytosol compared to the ER of HeLa cells, and identify the formation of oxidative oligomers of the sensors in the ER. Finally, we use new methodology to re-evaluate the binding parameters of these sensors both in the test tube and in living cells. Ultimately, we demonstrate that sensor responses can be affected by different cellular environments, and provide a framework for evaluating future generations of organelle-targeted sensors.

Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Native and engineered sensors for Ca2+ and Zn2+: lessons from calmodulin and MTF1.

Ca2+ and Zn2+ dynamics have been identified as important drivers of physiological processes. In order for these dynamics to encode function, the cell must have sensors that transduce changes in metal concentration to specific downstream actions. Here we compare and contrast the native metal sensors: calmodulin (CaM), the quintessential Ca2+ sensor and metal-responsive transcription factor 1 (MTF1), a candidate Zn2+ sensor. While CaM recognizes and modulates the activity of hundreds of proteins through allosteric interactions, MTF1 recognizes a single DNA motif that is distributed throughout the genome regulating the transcription of many target genes. We examine how the different inorganic chemistries of these two metal ions may shape these different mechanisms transducing metal ion concentration into changing physiologic activity. In addition to native metal sensors, scientists have engineered sensors to spy on the dynamic changes of metals in cells. The inorganic chemistry of the metals shapes the possibilities in the design strategies of engineered sensors. We examine how different strategies to tune the affinities of engineered sensors mirror the strategies nature developed to sense both Ca2+ and Zn2+ in cells.

Droplet Microfluidic Flow Cytometer For Sorting On Transient Cellular Responses Of Genetically-Encoded Sensors.

Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically encoded Zn2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.

Techniques for measuring cellular zinc.

The development and improvement of fluorescent Zn2+ sensors and Zn2+ imaging techniques have increased our insight into this biologically important ion. Application of these tools has identified an intracellular labile Zn2+ pool and cultivated further interest in defining the distribution and dynamics of labile Zn2+. The study of Zn2+ in live cells in real time using sensors is a powerful way to answer complex biological questions. In this review, we highlight newly engineered Zn2+ sensors, methods to test whether the sensors are accessing labile Zn2+, and recent studies that point to the challenges of using such sensors. Elemental mapping techniques can complement and strengthen data collected with sensors. Both mass spectrometry-based and X-ray fluorescence-based techniques yield highly specific, sensitive, and spatially resolved snapshots of metal distribution in cells. The study of Zn2+ has already led to new insight into all phases of life from fertilization of the egg to life-threatening cancers. In order to continue building new knowledge about Zn2+ biology it remains important to critically assess the available toolset for this endeavor.

Development of an Optical Zn2+ Probe Based on a Single Fluorescent Protein.

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering, and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1, which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). This sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.

Isothermal Titration Calorimetry Measurements of Metal Ions Binding to Proteins.

ITC measurements involving metal ions are susceptible to a number of competing reactions (oxidation, precipitation, and hydrolysis) and coupled reactions involving the buffer and protons. Stabilization and delivery of the metal ion as a well-defined and well-characterized complex with the buffer, or a specific ligand, can suppress undesired solution chemistry and, depending on the stability of the metal complex, allow accurate measurements of higher affinity protein-binding sites. This requires, however, knowledge of the thermodynamics of formation of the metal complex and accounting for its contribution to the experimentally measured values (KITC and ΔHITC) through a post hoc analysis that provides the condition-independent binding thermodynamics (K, ΔG(o), ΔH, ΔS, and ΔCP). This analysis also quantifies the number of protons that are displaced when the metal ion binds to the protein.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

BACKGROUND: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. RESULTS: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. CONCLUSION: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III.

Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato.

Comparative investigation of the reaction mechanisms of the organophosphate-degrading phosphotriesterases from Agrobacterium radiobacter (OpdA) and Pseudomonas diminuta (OPH).

Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.

Thermodynamics of formation of the insulin hexamer: metal-stabilized proton-coupled assembly of quaternary structure.

The thermodynamics of formation of the insulin hexamer, which is stabilized by two Zn(2+) ions, were quantified by isothermal titration calorimetry (ITC). Because the insulin monomer is unstable to aggregation (fibrillation) during ITC measurements, an original method involving EDTA chelation of Zn(2+) from the hexamer was employed. The two metal ions are chelated sequentially, reflecting stepwise Zn(2+) binding and stabilization of the quaternary structure. Analysis of the ITC data reveals that two to three H(+) bind to the hexamer upon its formation at pH 7.4, which is both enthalpically and entropically favored. The former is due to Zn(2+) coordination to His residues from three subunits, and the latter is associated with desolvation that accompanies the protonation and the packing of the subunits in the hexamer.

A mass spectrometric method for quantifying C3 and C6 phosphorylation of starch.

The glucosyl residues comprising starch can be phosphorylated at either the C3 or the C6 position of the molecule because of the activities of two distinct dikinase enzymes. After hydrolysis of the starch, the C6 phosphorylation is easy to measure using a routine enzyme assay for glucose 6-phosphate, but the C3 phosphorylation is more difficult to assay. A mass spectrometric (MS) method has been developed that, in a single run, can distinguish and quantify the glucose 3-phosphate and glucose 6-phosphate produced by hydrolysis of starch and can also measure the glucose content to give an accurate estimate of the starting material. The MS method involves quantification by LC/MS with external standards, using normal-phase hydrophilic interaction liquid chromatography and selective reaction monitoring. The MS method has been used to determine degrees of starch phosphorylation in a diverse group of potato lines, revealing threefold differences in phosphorylation between high- and low-phosphate lines. The method was also used to show that cold storage of potato tubers for up to 24weeks had little substantive effect on the levels of starch phosphorylation. MS provided an effective and efficient means of determining both the C6 and the C3 phosphorylation of starch.