Chloroplast biogenesis 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry

Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction. In this undertaking, Arabidopsis mutants that accumulate DV chlorophyllide a and/or DV chlorophyll [Chl(ide)] a are likely to provide an appropriate resource. Because the Arabidopsis genome has been completely sequenced, the best strategy for identifying 4VR and/or putative regulatory 4VR genes is to screen Arabidopsis Chl mutants for DV Chl(ide) a accumulation. In wild-type Arabidopsis, a DV plant species, only MV chlorophyllide (Chlide) a is detectable. However in Chl mutants lacking 4VR activity, DV Chl(ide) a may accumulate in addition to MV Chl(ide) a. In the current work, an in situ assay of DV Chl(ide) a accumulation, suitable for screening a large number of mutants lacking 4-vinyl Chlide a reductase activity with minimal experimental handling, is described. The assay involves homogenization of the tissues in Tris-HCl:glycerol buffer and the recording of Soret excitation spectra at 77K. DV Chlide a formation is detected by a Soret excitation shoulder at 459 nm over a wide range of DV Chlide a/MV Chl a ratios. The DV Chlide a shoulder became undetectable at DV Chlide a/MV Chl a ratios less than 0.049, that is, at a DV Chlide a content of less than 5%.     

Molecular phylogenetics of the melyrid lineage (Coleoptera: Cleroidea)

The melyrid lineage of beetles form a distinct group of the superfamily Cleroidea with a high level of soft‐bodiedness. Here we present the first molecular phylogenetic analysis of this group. The data matrix included partial sequences of the small and large subunits of rRNA, the mitochondrial large subunit rRNA, and cytochrome oxidase subunit I of 67 melyrid and eight outgroup taxa. The concatenated sequences were analysed using maximum‐parsimony (MP), maximum‐likelihood (ML) and Bayesian analysis (BA) approach. The results strongly supported the monophyly of the melyrid lineage splitting into six major clades: Rhadalidae, Mauroniscidae, Prionoceridae, Melyridae sensu stricto, Dasytidae and Malachiidae. The rhadalids were placed in the most basal position, followed by mauroniscids and prionocerids. Three terminal lineages—the true melyrids, dasytids, and malachiids—are well supported by all analyses, but their mutual relationships remain uncertain as MP analysis proposed alternative topologies to that of the ML and BA trees, with often low node support in the latter two methods. The monophyly of the subfamily Danacaeinae (Dasytidae) with respect to the danacaeine genera of the southern hemisphere (Hylodanacaea, Listrocerus, Amecocerus) was challenged as they were found to be polyphyletic. Similarly, the monophyly of Attalus was rejected by our analyses and shown to be polyphyletic. Based on the preferred phylogenetic hypothesis, the subfamilies Rhadalinae, Dasytinae and Malachiinae are elevated to family rank. © The Willi Hennig Society 2011.     

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/<Emphasis Type="Italic">neu</Emphasis>-tolerant mice

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT ⁺ (neuT ⁺) triple transgenic mice represent an improvement over neuT ⁺ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85–94) (p85) CTL and HER-2(776–790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT ⁺ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8⁺ CTLs and CD4⁺ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4⁺ and CD8⁺ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4⁺ and CD8⁺ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.     

Use of SANS and biophysical techniques to reveal subtle conformational differences between native apo-calmodulin and its unfolded states

Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85 °C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin.     

In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species

Efficient micropropagation and cryopreservation of Hypericum richeri ssp. transsilvanicum, an endemic species in Romania, and Hypericum umbellatum, a rare and endangered Daco-Balkan species, was achieved. The effects of type of explant and cytokinin on in vitro plant regeneration were investigated. Shoot organogenesis was achieved in all explants, but stem nodes regenerated best. Organogenesis from nodal segments was promoted by incubating these explants on Murashige and Skoog (MS) medium in the presence of cytokinins (6-benzyladenine, thidiazuron, kinetin or 6-??,??-dimethylallylaminopurine), each tested at four concentrations. The best morphogenic response for both Hypericum species (number of shoots per explant, shoot length, axillary branching of shoot, and frequency of shoot organogenesis) was observed when explants were incubated on MS medium containing 0.44 or 1.11???M 6-benzyladenine. Root induction was achieved only when regenerated shoots were transferred to fresh medium with or without auxin. Maximum rooting was recorded on MS medium supplemented with 2.45???M indole-3-butyric acid. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development. Shoot tips and axillary buds excised from the in vitro regenerated plants were successfully cryopreserved in liquid nitrogen by the droplet-vitrification method. Following preculture in 0.25?M sucrose, dehydration and cryopreservation, the highest regeneration rates were obtained in both species by using axillary buds (68?% for H. richeri ssp. transsilvanicum and 71?% for H. umbellatum).     

Unfakeable facial configurations affect strategic choices in trust games with or without information about past behavior

Background

Many human interactions are built on trust, so widespread confidence in first impressions generally favors individuals with trustworthy-looking appearances. However, few studies have explicitly examined: 1) the contribution of unfakeable facial features to trust-based decisions, and 2) how these cues are integrated with information about past behavior.

Methodology/Principal Findings

Using highly controlled stimuli and an improved experimental procedure, we show that unfakeable facial features associated with the appearance of trustworthiness attract higher investments in trust games. The facial trustworthiness premium is large for decisions based solely on faces, with trustworthy identities attracting 42% more money (Study 1), and remains significant though reduced to 6% when reputational information is also available (Study 2). The face trustworthiness premium persists with real (rather than virtual) currency and when higher payoffs are at stake (Study 3).

Conclusions/Significance

Our results demonstrate that cooperation may be affected not only by controllable appearance cues (e.g., clothing, facial expressions) as shown previously, but also by features that are impossible to mimic (e.g., individual facial structure). This unfakeable face trustworthiness effect is not limited to the rare situations where people lack any information about their partners, but survives in richer environments where relevant details about partner past behavior are available.     

Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis

Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE(-/-) mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE(-/-) mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE(-/-) mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α(1)-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α(1)-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE(-/-) mice on HFD. Urinary excretion levels of collagen and α(1)-antitrypsin fragments also significantly correlated with intraplaque collagen and α(1)-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α(1)-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.     

Sequential feeding with variations in energy and protein levels improves gait score in meat-type chickens

Feeding broilers by alternating different diets for 1 or 2 days is known as sequential feeding, and it possibly reduces leg problems since it slows down early growth and may enhance general activity. The present study compared continuous feeding with a standard diet (C: metabolisable energy = 12.55 MJ/kg, crude protein = 190 g/kg) with alternations of a high-energy/low-protein diet (E+P-:+7% ME; -20% CP) and a low-energy/high-protein diet (E-P+: -7% ME,+20% CP) and investigated its effects on growth, behaviour and gait score in 352 male Ross broiler chickens. Sequential feeding was carried out during ten 48-h sequential-feeding cycles from 8 to 28 days of age. Three treatments were compared: complete diet (C) and two alternations of diets varying in protein and energy contents (S1: E+P- followed by E-P+; and S2: E-P+ followed by E+P-). Chickens received the same feed during the starter and finisher periods (0 to 7 and 29 to 38 days of age). Body weight (BW), feed intake, general activity and gait score, bone quality and carcass conformation were measured to evaluate leg condition and general performance. Sequential feeding significantly reduced BW at 28 days of age (S1: -9.1%; S2: -3.7%/C group; P < 0.05) and S1 were lighter than S2. In both sequential groups, time spent standing increased (C: 28%; S1:33%; S2: 35%; P < 0.05) and leg abnormalities decreased (mean gait score: C: 2.61; S1: 2.45; S2: 2.38; P < 0.02). This improvement was not related to changes in bone quality. BW at slaughter was impaired in Group S1 only, and the feed conversion ratio throughout the rearing period was not significantly impaired by sequential feeding. However, abdominal fat was higher in the S2 group. Sequential feeding using diets varying in energy and crude protein can be a useful method of reducing leg problems in broilers since it improves gait score without impairing growth performance when used as early as 8 days of age and up to not less than 8 days before slaughter in order to compensate for reduced growth. This improvement can be explained by reduced early growth and enhanced motor activity. However, it appears that the low-energy diet should be given first in order to avoid a reduction in BW at slaughter.     

Sampling-Based Approaches to Improve Estimation of Mortality among Patient Dropouts: Experience from a Large PEPFAR-Funded Program in Western Kenya

Background

Monitoring and evaluation (M&E) of HIV care and treatment programs is impacted by losses to follow-up (LTFU) in the patient population. The severity of this effect is undeniable but its extent unknown. Tracing all lost patients addresses this but census methods are not feasible in programs involving rapid scale-up of HIV treatment in the developing world. Sampling-based approaches and statistical adjustment are the only scaleable methods permitting accurate estimation of M&E indices.

Methodology/Principal Findings

In a large antiretroviral therapy (ART) program in western Kenya, we assessed the impact of LTFU on estimating patient mortality among 8,977 adult clients of whom, 3,624 were LTFU. Overall, dropouts were more likely male (36.8% versus 33.7%; p = 0.003), and younger than non-dropouts (35.3 versus 35.7 years old; p = 0.020), with lower median CD4 count at enrollment (160 versus 189 cells/ml; p<0.001) and WHO stage 3–4 disease (47.5% versus 41.1%; p<0.001). Urban clinic clients were 75.0% of non-dropouts but 70.3% of dropouts (p<0.001). Of the 3,624 dropouts, 1,143 were sought and 621 had their vital status ascertained. Statistical techniques were used to adjust mortality estimates based on information obtained from located LTFU patients. Observed mortality estimates one year after enrollment were 1.7% (95% CI 1.3%–2.0%), revised to 2.8% (2.3%–3.1%) when deaths discovered through outreach were added and adjusted to 9.2% (7.8%–10.6%) and 9.9% (8.4%–11.5%) through statistical modeling depending on the method used. The estimates 12 months after ART initiation were 1.7% (1.3%–2.2%), 3.4% (2.9%–4.0%), 10.5% (8.7%–12.3%) and 10.7% (8.9%–12.6%) respectively.

Conclusions/Significance Abstract

Assessment of the impact of LTFU is critical in program M&E as estimated mortality based on passive monitoring may underestimate true mortality by up to 80%. This bias can be ameliorated by tracing a sample of dropouts and statistically adjust the mortality estimates to properly evaluate and guide large HIV care and treatment programs.     

Correlation of low molecular weight advanced glycation end products and nitric oxide metabolites with chronic complications in type 1 diabetic patients

Advanced glycation end products (AGEs) are involved in the occurrence of vascular complications in diabetes. The present study was undertaken to investigate the level of low-molecular weight products of AGEs (LMW-AGEs) in relation to microvascular complications in type 1 diabetes, and the possible relationship with nitric oxide (NO) as a marker of endothelial function. Patients with normal renal function (NRF) were classified into two groups: (1) without, and (2) with diabetic neuropathy; and patients with renal impairment also into two groups: (3) diabetic renal disease, and (4) end-stage renal disease. The fluorescence of LMW-AGEs and measurement of NO metabolites was assessed in 277 serum samples. In addition, multiple regression analysis was performed. In group 1, LMW-AGEs level (9.3±1.1 AF%) was higher than in the control group (2.4±0.3 AF%). A trend in the increase of LMW-AGEs with neuropathy (29.7±5.5 AF%, group 2), and further with renal impairment (47.0±8.0, group 3 and 137.8±25.5 AF%, group 4), was observed. In multivariate regression analysis LMW-AGEs were associated with NO metabolites in group 2. In NRF patients, diabetic neuropathy was significantly correlated with LMW-AGEs and NO metabolites, independently of serum creatinine and duration of diabetes. This relationship suggests that the NO and LMW-AGEs’ actions (possibly synergistic) in endothelial activation possess a role in the initiation and development of diabetic microvascular complications.