What's in a name; Genetic structure in Solanum section Petota studied using population-genetic tools

Background  

The taxonomy and systematic relationships among species of Solanum section Petota are complicated and the section seems overclassified. Many of the presumed (sub)species from South America are very similar and they are able to exchange genetic material. We applied a population genetic approach to evaluate support for subgroups within this material, using AFLP data. Our approach is based on the following assumptions: (i) accessions that may exchange genetic material can be analyzed as if they are part of one gene pool, and (ii) genetic differentiation among species is expected to be higher than within species.     

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.     

Inhibition of the HIV-1 Spike by Single-PG9/16-Antibody Binding Suggests a Coordinated-Activation Model for Its Three Protomeric Units

The HIV-1 spike is composed of three protomeric units, each containing a peripheral gp120 and a transmembrane gp41 subunit. Binding to the CD4 and the chemokine receptors triggers them to mediate virus entry into cells by membrane fusion. The spikes also represent the major target for neutralizing antibodies (Abs) against the virus. We have studied how two related broadly neutralizing Abs, PG9 and PG16, react with the spike. Unexpectedly, this also suggested how the functions of the individual protomers in the spike depend on each other. The Abs have been shown to bind the V1/V2 loops of gp120, located at the top of the spike. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we show that only single Abs or antigen-binding fragments could bind to the spikes of HIV-1 virus-like particles. Apparently, binding to one gp120 sterically interferes with binding to the other two subunits in the spike top. Despite this constraint, all of the protomers of the spike became resistant to CD4 binding and subsequent formation of the coreceptor binding site. These activities were measured by monitoring the sequential complex formation of the spike first with Abs and then with soluble 2d- or 4d-CD4 or with soluble CD4 and the CD4 inducible coreceptor binding site Ab 17b in BN-PAGE. The inhibition of the spike by single-Ab binding suggested that the activation reactions of the individual protomeric units are linked to each other in a coordinated activation process.     

A Human Antibody to the CD4 Binding Site of gp120 Capable of Highly Potent but Sporadic Cross Clade Neutralization of Primary HIV-1

Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.     

Predicting expected progeny difference for marbling score in Angus cattle using artificial neural networks and Bayesian regression models

Background

Artificial neural networks (ANN) mimic the function of the human brain and are capable of performing massively parallel computations for data processing and knowledge representation. ANN can capture nonlinear relationships between predictors and responses and can adaptively learn complex functional forms, in particular, for situations where conventional regression models are ineffective. In a previous study, ANN with Bayesian regularization outperformed a benchmark linear model when predicting milk yield in dairy cattle or grain yield of wheat. Although breeding values rely on the assumption of additive inheritance, the predictive capabilities of ANN are of interest from the perspective of their potential to increase the accuracy of prediction of molecular breeding values used for genomic selection. This motivated the present study, in which the aim was to investigate the accuracy of ANN when predicting the expected progeny difference (EPD) of marbling score in Angus cattle. Various ANN architectures were explored, which involved two training algorithms, two types of activation functions, and from 1 to 4 neurons in hidden layers. For comparison, BayesCπ models were used to select a subset of optimal markers (referred to as feature selection), under the assumption of additive inheritance, and then the marker effects were estimated using BayesCπ with π set equal to zero. This procedure is referred to as BayesCpC and was implemented on a high-throughput computing cluster.

Results

The ANN with Bayesian regularization method performed equally well for prediction of EPD as BayesCpC, based on prediction accuracy and sum of squared errors. With the 3K-SNP panel, for example, prediction accuracy was 0.776 using BayesCpC, and ranged from 0.776 to 0.807 using BRANN. With the selected 700-SNP panel, prediction accuracy was 0.863 for BayesCpC and ranged from 0.842 to 0.858 for BRANN. However, prediction accuracy for the ANN with scaled conjugate gradient back-propagation was lower, ranging from 0.653 to 0.689 with the 3K-SNP panel, and from 0.743 to 0.793 with the selected 700-SNP panel.

Conclusions

ANN with Bayesian regularization performed as well as linear Bayesian regression models in predicting additive genetic values, supporting the idea that ANN are useful as universal approximators of functions of interest in breeding contexts.     

Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals

A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 "tier 2" viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.     

EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat

Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.     

Meta-analysis identified the TNFA -308G > A promoter polymorphism as a risk factor for disease severity in patients with rheumatoid arthritis

Introduction

The goal of this study is to investigate whether the -308G > A promoter polymorphism in the tumor necrosis factor alpha (TNFA) gene is associated with disease severity and radiologic joint damage in a large cohort of patients with rheumatoid arthritis (RA).

Methods

A long-term observational early RA inception cohort (n = 208) with detailed information about disease activity and radiologic damage after 3, 6 and 9 years of disease was genotyped for the TNFA -308G > A promoter polymorphism (rs1800629). A longitudinal regression analysis was performed to assess the effect of genotype on RA disease severity and joint damage. Subsequently, a meta-analysis, including all publically available data, was performed to further test the association between joint erosions and the TNFA polymorphism. To learn more about the mechanism behind the effect of the polymorphism, RNA isolated from peripheral blood from RA patients (n = 66) was used for TNFA gene expression analysis by quantitative PCR.

Results

Longitudinal regression analysis with correction for gender and disease activity showed a significant difference in total joint damage between GG and GA+AA genotype groups (P = 0.002), which was stable over time. The meta-analysis, which included 2,053 patients, confirmed an association of the genetic variant with the development of erosions (odds ratio 0.78, 95% CI 0.62, 0.98). No significant differences in TNFA gene expression were observed for the different genotypes, confirming earlier findings in healthy individuals.

Conclusions

Our data confirm that the TNFA -308G > A promoter polymorphism is associated with joint damage in patients with RA. This is not mediated by differences in TNFA gene expression between genotypes.     

Prediction and course of symptoms and lung function around an exacerbation in chronic obstructive pulmonary disease

Background

Frequent exacerbations induce a high burden to Chronic Obstructive Pulmonary Disease (COPD). We investigated the course of exacerbations in the published COSMIC study that investigated the effects of 1-year withdrawal of fluticasone after a 3-month run-in treatment period with salmeterol/fluticasone in patients with COPD.

Methods

In 373 patients, we evaluated diary cards for symptoms, Peak Expiratory Flow (PEF), and salbutamol use and assessed their course during exacerbations.

Results

There were 492 exacerbations in 224 patients. The level of symptoms of cough, sputum, dyspnea and nocturnal awakening steadily increased from 2 weeks prior to exacerbation, with a sharp rise during the last week. Symptoms of cough, sputum, and dyspnea reverted to baseline values at different rates (after 4, 4, and 7 weeks respectively), whereas symptoms of nocturnal awakening were still increased after eight weeks. The course of symptoms was similar around a first and second exacerbation. Increases in symptoms and salbutamol use and decreases in PEF were associated with a higher risk to develop an exacerbation, but with moderate predictive values, the areas under the receiver operating curves ranging from 0.63 to 0.70.

Conclusions

Exacerbations of COPD are associated with increased symptoms that persist for weeks and the course is very similar between a first and second exacerbation. COPD exacerbations are preceded by increased symptoms and salbutamol use and lower PEF, yet predictive values are too low to warrant daily use in clinical practice.