Voltage regulation of single green fluorescent protein mutants

We report the analysis of the fluorescence intensity fluctuations of single proteins of a GFP mutant, GFPmut2, embedded in a polyelectrolyte nanocapsule adsorbed on thin conductive layers. Our results, based on single molecule fluorescence spectroscopy, indicate that the fluorescence blinking dynamics of GFP is strongly dependent on the bulk conductivity of the metal layer substrate, on the distance from the conductive surfaces and on the amplitude of the voltage applied to the poly-electrolyte layers. These findings suggest that fluorescence blinking itself might be employed as a reporter signal in nano-bio-technology applications.     

Seasonal changes in digestive enzyme (trypsin) activity of the copepods <Emphasis Type="Italic">Pseudocalanus minutus</Emphasis> (Calanoida) and <Emphasis Type="Italic">Oithona similis</Emphasis> (Cyclopoida) in the Arctic Kongsfjorden (Svalbard)

Seasonal activities of the digestive enzyme trypsin were measured between August 1998 and May 1999 to study different nutritional strategies of the two copepods Pseudocalanus minutus and Oithona similis in the Arctic Kongsfjorden (Svalbard) using a highly sensitive fluorescence technique. Stage-, depth- and season-specific characteristics of digestive activity were reflected in the trypsin activity. P. minutus females and stage V copepodids (C) had highest trypsin activities in spring during reproduction (197.5 and 145.7 nmol min⁻¹ ng C⁻¹, respectively). In summer stages CIII–V and in autumn stages CIV and V had high activities (80–116 nmol min⁻¹ ng C⁻¹) in the shallow layer (< 100 m) presumably as a consequence of prolonged feeding before descending to overwintering depth. Trypsin activities at depth (> 100 m) in summer and autumn were low in stages CIII and CIV (29–60 nmol min⁻¹ ng C⁻¹) and in winter in all stages in both layers (20–43 nmol min⁻¹ ng C⁻¹). Based on low trypsin activity, males most likely did not feed. In O. similis, the spring phytoplankton bloom did not significantly affect trypsin activity as compared to the other seasons. O. similis CV and females had high trypsin activities in summer in the deep stratum (304.5 nmol min⁻¹ ng C⁻¹), which was concomitant with reproductive processes and energy storage for overwintering. In autumn, stage CV and female O. similis had significantly higher activities than stage CIV (130–152 versus 78 nmol min⁻¹ ng C⁻¹), which is in accordance with still ongoing developmental and reproductive processes in CVs and females. Comparisons of both species revealed different depth-related responses emphasizing different nutritional preferences: the mainly herbivorous P. minutus is more actively feeding in the shallow layer, where primary production occurs, whereas the omnivorous O. similis is not as much restricted to a certain depth layer, when searching for food. P. minutus had lower levels of trypsin activity during all seasons. In contrast to P. minutus, higher enzyme activities in males of O. similis suggest that they continue to feed and survive after fertilization of females.     

Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry

In general, mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). Here we introduce high-resolution tandem mass spectrometry (quadrupole-Orbitrap) for the quantification of 25-hydroxy-vitamin D [25(OH)D], a marker of the vitamin D status, because the specificity of 25(OH)D immunoassays is still questionable and mass spectrometric quantification is becoming increasingly important. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) was used to quantify 25-hydroxy-cholecalciferol [25(OH)D₃], 25-hydroxy-ergocalciferol [25(OH)D₂], and their C3-epimers 3-epi-25(OH)D₃ and 3-epi-25(OH)D₂. The method has a run time of 5 min and was validated according to the US Food and Drug Administration and the European Medicines Agency guidelines. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D₆-25(OH)D₂ with 3-epi-25(OH)D₃. All analytes showed an imprecision of below 10% coefficient of variation (CV), trueness between 90% and 110%, and limits of quantification below 10 nM. Concentrations measured by LC-MS/HR-MS are in good agreement with those of the National Institute of Standards and Technology reference methods using LC-MS/MS (QQQ). In conclusion, quantification of 25(OH)D by LC-MS/HR-MS is applicable for routine testing and also holds promise for highly specific quantification of other small molecules.     

Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes

Expression of the β-subunit (Ca_Vβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. Ca_Vβ binds to the α₁ pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although Ca_Vβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between Ca_Vβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by Ca_Vβ₂ that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of Ca_Vβ₂-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. Ca_Vβ mediated L-type up-regulation, and Ca_Vβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of Ca_Vβ that is directly involved in vesicular trafficking. We propose a model in which Ca_Vβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane.     

A Systematic Review and Meta-Analysis on the Safety of Vascular Endothelial Growth Factor (VEGF) Inhibitors for the Treatment of Retinopathy of Prematurity

Introduction

Laser photocoagulation is the current gold standard treatment for proliferative retinopathy of prematurity (ROP). However, it permanently reduces the visual field and might induce myopia. Vascular endothelial growth factor (VEGF) inhibitors for the treatment of ROP may enable continuing vascularization of the retina, potentially allowing the preservation of the visual field. However, for their use in infants concern remains. This meta-analysis explores the safety of VEGF inhibitors.

Methods

The Ovid Interface was used to perform a systematic review of the literature in the databases PubMed, EMBASE and the Cochrane Library.

Results

This meta-analysis included 24 original reports (including 1.457 eyes) on VEGF inhibitor treatment for ROP. The trials were solely observational except for one randomized and two case-control studies. We estimated a 6-month risk of retreatment per eye of 2.8%, and a 6-month risk of ocular complication without the need of retreatment of 1.6% per eye. Systemic complications were only reported as isolated incidents.

Discussion

VEGF inhibitors seem to be associated with low recurrence rates and ocular complication rates. They may have the benefit of potentially allowing the preservation of visual field and lower rates of myopia. Due to the lack of data, the risk of systemic side effects cannot be assessed.     

A Computational Gene Expression Score for Predicting Immune Injury in Renal Allografts

Background

Whole genome microarray meta-analyses of 1030 kidney, heart, lung and liver allograft biopsies identified a common immune response module (CRM) of 11 genes that define acute rejection (AR) across different engrafted tissues. We evaluated if the CRM genes can provide a molecular microscope to quantify graft injury in acute rejection (AR) and predict risk of progressive interstitial fibrosis and tubular atrophy (IFTA) in histologically normal kidney biopsies.

Methods

Computational modeling was done on tissue qPCR based gene expression measurements for the 11 CRM genes in 146 independent renal allografts from 122 unique patients with AR (n = 54) and no-AR (n = 92). 24 demographically matched patients with no-AR had 6 and 24 month paired protocol biopsies; all had histologically normal 6 month biopsies, and 12 had evidence of progressive IFTA (pIFTA) on their 24 month biopsies. Results were correlated with demographic, clinical and pathology variables.

Results

The 11 gene qPCR based tissue CRM score (tCRM) was significantly increased in AR (5.68 ± 0.91) when compared to STA (1.29 ± 0.28; p < 0.001) and pIFTA (7.94 ± 2.278 versus 2.28 ± 0.66; p = 0.04), with greatest significance for CXCL9 and CXCL10 in AR (p <0.001) and CD6 (p<0.01), CXCL9 (p<0.05), and LCK (p<0.01) in pIFTA. tCRM was a significant independent correlate of biopsy confirmed AR (p < 0.001; AUC of 0.900; 95% CI = 0.705–903). Gene expression modeling of 6 month biopsies across 7/11 genes (CD6, INPP5D, ISG20, NKG7, PSMB9, RUNX3, and TAP1) significantly (p = 0.037) predicted the development of pIFTA at 24 months.

Conclusions

Genome-wide tissue gene expression data mining has supported the development of a tCRM-qPCR based assay for evaluating graft immune inflammation. The tCRM score quantifies injury in AR and stratifies patients at increased risk of future pIFTA prior to any perturbation of graft function or histology.     

Associations between Meteorological Parameters and Influenza Activity in Berlin (Germany), Ljubljana (Slovenia), Castile and León (Spain) and Israeli Districts

Background

Studies in the literature have indicated that the timing of seasonal influenza epidemic varies across latitude, suggesting the involvement of meteorological and environmental conditions in the transmission of influenza. In this study, we investigated the link between meteorological parameters and influenza activity in 9 sub-national areas with temperate and subtropical climates: Berlin (Germany), Ljubljana (Slovenia), Castile and León (Spain) and all 6 districts in Israel.

Methods

We estimated weekly influenza-associated influenza-like-illness (ILI) or Acute Respiratory Infection (ARI) incidence to represent influenza activity using data from each country’s sentinel surveillance during 2000–2011 (Spain) and 2006–2011 (all others). Meteorological data was obtained from ground stations, satellite and assimilated data. Two generalized additive models (GAM) were developed, with one using specific humidity as a covariate and another using minimum temperature. Precipitation and solar radiation were included as additional covariates in both models. The models were adjusted for previous weeks’ influenza activity, and were trained separately for each study location.

Results

Influenza activity was inversely associated (p<0.05) with specific humidity in all locations. Minimum temperature was inversely associated with influenza in all 3 temperate locations, but not in all subtropical locations. Inverse associations between influenza and solar radiation were found in most locations. Associations with precipitation were location-dependent and inconclusive. We used the models to estimate influenza activity a week ahead for the 2010/2011 period which was not used in training the models. With exception of Ljubljana and Israel’s Haifa District, the models could closely follow the observed data especially during the start and the end of epidemic period. In these locations, correlation coefficients between the observed and estimated ranged between 0.55 to 0.91and the model-estimated influenza peaks were within 3 weeks from the observations.

Conclusion

Our study demonstrated the significant link between specific humidity and influenza activity across temperate and subtropical climates, and that inclusion of meteorological parameters in the surveillance system may further our understanding of influenza transmission patterns.     

Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.     

Differential gene expression after zinc supplementation and deprivation in human leukocyte subsets

An individual's zinc status has a significant impact on the immune system, and zinc deficiency, as well as supplementation, modulates immune function. To investigate the effects of zinc on different leukocyte subsets, we used microarray technology to analyze and compare the changes in mRNA expression in cell culture models of monocytes (THP-1), T cells (Jurkat), and B cells (Raji), in response to supplementation for 40 h with 50 microM zinc or 2.5 microM of the membrane-permeant zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine], respectively. In each cell type, several hundred genes were identified to be zinc sensitive, but only a total of seven genes were commonly regulated in all three cell lines. The majority of those genes were involved in zinc homeostasis, and none in immune function. Nevertheless, further analysis revealed that zinc affects entire functional networks of genes that are related to proinflammatory cytokines and cellular survival. Although the zinc-regulated activities are similar throughout the gene networks, the specific genes that are affected vary significantly between different cell types, a situation that helps to elucidate the disparity of the effects that zinc has on different leukocyte populations.