Use of national and international growth charts for studying height in European children: development of up-to-date European height-for-age charts

Background

Growth charts based on data collected in different populations and time periods are key tools to assess children’s linear growth. We analyzed the impact of geographic factors and the secular trend on height-for-age charts currently used in European populations, developed up-to-date European growth charts, and studied the effect of using different charts in a sample of growth retarded children.

Methods and Findings

In an international survey we obtained 18 unique national height-for-age charts from 28 European countries and compared them with charts from the World Health Organization (WHO), Euro-Growth reference, and Centers of Disease Control and Prevention (CDC). As an example, we obtained height data from 3,534 children with end-stage renal disease (ESRD) from 13 countries via the ESPN/ERA-EDTA registry, a patient group generally suffering from growth retardation. National growth charts showed a clear secular trend in height (mean height increased on average 0.6 cm/decade) and a North-South height gradient in Europe. For countries without a recent (>1990) national growth chart novel European growth charts were constructed from Northern and Southern European reference populations, reflecting geographic height differences in mean final height of 3.9 cm in boys and 3.8 cm in girls. Mean height SDS of 2- to 17-year-old ESRD patients calculated from recent national or derived European growth charts (−1.91, 95% CI: −1.97 to −1.85) was significantly lower than when using CDC or WHO growth charts (−1.55, 95% CI: −1.61 to −1.49) (P<0.0001).

Conclusion

Differences between height-for-age charts may reflect true population differences, but are also strongly affected by the secular trend in height. The choice of reference charts substantially affects the clinical decision whether a child is considered short-for-age. Therefore, we advocate using recent national or European height-for-age charts derived from recent national data when monitoring growth of healthy and diseased European children.     

Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days

Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.     

Improvement of tuberculosis laboratory capacity on pemba island, zanzibar: a health cooperation project

Low-income countries with high Tuberculosis burden have few reference laboratories able to perform TB culture. In 2006, the Zanzibar National TB Control Programme planned to decentralize TB diagnostics. The Italian Cooperation Agency with the scientific support of the "L. Spallanzani" National Institute for Infectious Diseases sustained the project through the implementation of a TB reference laboratory in a low-income country with a high prevalence of TB. The implementation steps were: 1) TB laboratory design according to the WHO standards; 2) laboratory equipment and reagent supplies for microscopy, cultures, and identification; 3) on-the-job training of the local staff; 4) web- and telemedicine-based supervision.From April 2007 to December 2010, 921 sputum samples were received from 40 peripheral laboratories: 120 TB cases were diagnosed. Of all the smear-positive cases, 74.2% were culture-positive. During the year 2010, the smear positive to culture positive rate increased up to 100%.In March 20, 2010 the Ministry of Health and Social Welfare of Zanzibar officially recognized the Public Health Laboratory- Ivo de Carneri as the National TB Reference Laboratory for the Zanzibar Archipelago.An advanced TB laboratory can represent a low cost solution to strengthen the TB diagnosis, to provide capacity building and mid-term sustainability.     

Structural and functional analysis of the archaeal endonuclease Nob1

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.     

The mitochondrial permeability transition pore (PTP) — An example of multiple molecular exaptation?

The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500 Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins.In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role.Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.     

Cellular stress responses, hormetic phytochemicals and vitagenes in aging and longevity

Modulation of endogenous cellular defense mechanisms represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as in neurodegeneration. This paper introduces the emerging role of exogenous molecules in hormetic-based neuroprotection and the mitochondrial redox signaling concept of hormesis and its applications to the field of neuroprotection and longevity. Maintenance of optimal long-term health conditions is accomplished by a complex network of longevity assurance processes that are controlled by vitagenes, a group of genes involved in preserving cellular homeostasis during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. Dietary antioxidants, such as polyphenols and L-carnitine/acetyl-L-carnitine, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. Hormesis provides the central underpinning of neuroprotective responses, providing a framework for explaining the common quantitative features of their dose response relationships, their mechanistic foundations, their relationship to the concept of biological plasticity as well as providing a key insight for improving the accuracy of the therapeutic dose of pharmaceutical agents within the highly heterogeneous human population. This paper describes in mechanistic detail how hormetic dose responses are mediated for endogenous cellular defense pathways including sirtuin, Nrfs and related pathways that integrate adaptive stress responses in the prevention of neurodegenerative diseases. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Single-Particle Tracking Demonstrates that Actin Coordinates the Movement of the Ebola Virus Matrix Protein

The Ebola virus causes severe hemorrhagic fever and has a mortality rate that can be as high as 90%, yet no vaccines or approved therapeutics, to our knowledge, are available. To replicate and egress the infected host cell the Ebola virus uses VP40, its major matrix protein to assemble at the inner leaflet of the plasma membrane. The assembly and budding of VP40 from the plasma membrane of host cells seem still poorly understood. We investigated the assembly and egress of VP40 at the plasma membrane of human cells using single-particle tracking. Our results demonstrate that actin coordinates the movement and assembly of VP40, a critical step in viral egress. These findings underscore the ability of single-molecule techniques to investigate the interplay of VP40 and host proteins in viral replication.The actin cortex below the plasma membrane of mammalian cells is essential for maintenance of cell shape and for cell movement. This cortex has also been found to play an essential role in the replication process of a number of viruses including West Nile virus (1), respiratory syncytial virus (2), influenza (3), and vaccinia virus (4). Additionally, actin has been found to play a central role in the assembly and budding of HIV-1 (5) whereas Marburg virus has been shown to use actin-enriched filopodia to exit the host cell (6). Actin has also been found to be packaged into Ebola-virus-like particles (VLPs) (7). Ebola virus, which causes severe hemorrhagic fever, harbors a single-stranded negative-sense RNA genome encoding seven proteins. Of these seven proteins, VP40 is the most abundantly expressed and has been found to play a central role in the budding of the virus from the plasma membrane (8). Whereas actin has been found in Ebola VLPs (7), the role of actin in Ebola VP40 assembly is still seemingly unknown. Here, we have used Raster image correlation spectroscopy (RICS) (9) and three-dimensional single-particle tracking (see Fig. S1 in the Supporting Material) (10) to investigate the dynamics of Ebola VP40 and actin. We report that preassembled VLPs (pVLPs) of Ebola VP40 require actin for directed movement and assembly.Ebola VP40 has been demonstrated to colocalize with actin and actin is found in VP40 VLPs (7), suggesting an important role for actin in the replication cycle of the virus. To confirm the colocalization between VP40 and actin in HEK293 and CHO-K1 cells, we used confocal microscopy to examine the distribution of EGFP-VP40 and mCherry-actin. EGFP-VP40 and mCherry-actin displayed colocalization at the plasma membrane of HEK293 and CHO-K1 cells (see Fig. S2 A), which was markedly reduced in response to treatment with LAT-A (see Fig. S2 B and Fig. S3 A), an actin polymerization inhibitor. VP40 plasma membrane localization was not disrupted by LAT-A treatment (not unexpected, as VP40 is a lipid-binding protein (11) where high affinity for the PM drives its cellular localization (E. Adu-Gyamfi and R. V. Stahelin, unpublished)). To test whether this VP40-actin interaction is important to viral egress, we detected EGFP-VP40 with an anti-EGFP antibody used to measure VLPs formed from cells expressing EGFP-VP40. This was also performed to assess the effect of pharmacological treatment on EGFP-VP40-expressing cells with LAT-A or with the microtubule polymerization inhibitor nocodazole (see Fig. S3 B). LAT-A treatment led to a significant reduction in VLP formation whereas nocodazole did not display detectable effects.To test whether the VP40 and actin are engaged in synchronized movement, we performed time-lapse imaging in both the green and red channels. We observed that the pVLPs move with actin fibers extending from the plasma membrane (see Movie S1 in the Supporting Material). The movement was rapid, and caused smaller particles to merge into larger filamentous forms. To further demonstrate that the motion of actin and VP40 spatially overlapped, we used RICS to obtain correlation maps of EGFP-VP40 and mCherry-actin (Fig. 1). The spatial cross-correlation map indicated significant overlap of VP40 and actin movement (Fig. 2, A–C) at the plasma membrane (Fig. 1 and see Fig. S6), but not in the cytosol (see Fig. S5 and Fig. S7). In contrast, EGFP-VP40 and mCherry-α-tubulin (see Fig. S8, Fig. S9, and Fig. S10) displayed no significant spatial cross-correlation at the plasma membrane (Fig. S11) or other regions of the cell (see Fig. S12), supporting the VLP egress data where inhibition of microtubule polymerization did not influence viral egress.Open in a separate windowFigure 1EGFP-VP40 and mCherry-actin RICS analysis at the membrane. (A) HEK293 cells expressing EGFP-VP40 and mCherry-actin were imaged for 100 frames at 256 × 256 pixels. (White scale bar = 2 μm.) (B) Average intensity image of EGFP-VP40 across the 100 collected frames. (Pink box) Used to select a region of interest to yield the (C) average EGFP-VP40 intensity image. (D) Average intensity image of mCherry-actin taken for 100 frames at 256 × 256 pixels was used to select the same region of interest as in panel B (pink box) to yield the (E) average intensity image of the mCherry-actin signal in this region. (F) The two-dimensional spatial cross-correlation analysis of panels C and E demonstrates significant cross-correlation of VP40 and actin signals.Open in a separate windowFigure 2Three-dimensional RICS correlation maps of VP40 and actin cross-correlate at the plasma membrane. (A) EGFP-VP40 and (B) mCherry-actin (Fig. 1 and see Fig. S6 in the Supporting Material) RICS autocorrelation functions. (C) Appreciable cross-correlation is observed for EGFP-VP40 and mCherry-actin at the plasma membrane.To test whether the motion of the pVLPs is directed by actin, we applied the three-dimensional orbital tracking method first introduced by Levi et. al. (10). Tracking of isolated particles (Fig. 3 A) in five different cells allowed determination of the pVLPs trajectories (Fig. 3 D), which suggested that the VP40 particles undergo a directed motion. To verify this, we plotted the mean-square displacement (MSD) curves for the pVLPs (Fig. 3 C), which confirmed the trajectory was characteristic of directed motion. Analysis of the intensity profile of the dynamic VP40 particles suggested that the intensity of the particle changes with respect to time. Bleaching is expected if the molecule is exposed to the laser beam for an extended period of time; however, an increase in intensities was observed along the trajectory of the green channel due to addition of VP40 molecules. This suggests that the movement of the particles along actin fibers promote multimerization and maturation of the pVLPs. When actin polymerization was inhibited in four different cells with LAT-B, the rapid movement (see Fig. S13) and the directed trajectories of the pVLPs were lost (Fig. 3, E and F). This was reflected in a change from directed motion to movement indicative of random then constrained diffusion (Fig. 3, E and F).Open in a separate windowFigure 3Actin directs the movement of VP40 particles. HEK293 cells transfected with EGFP-VP40 were imaged with an electronic zoom of 2000 mV, corresponding to 72 nm/pixel in both X and Y. (A) An isolated and representative VP40 particle (highlighted by white box, inset) was tracked as described in the Supporting Material. (B) Intensity profile of the pVLP in A demonstrates increases in EGFP-VP40 intensity along the trajectory. (C) MSD of the pVLP, which follows a ballistic motion with a velocity of 0.067 ± 0.01 μm² s⁻¹. (D) The three-dimensional trajectory of the particle shown in panels A–C. (E) MSD curve of VP40 particles yields random then constrained diffusion after LAT-B treatment with a mean velocity of 0.017 ± 0.006 μm² s⁻¹ (p < 0.001). (F) Three-dimensional trajectory of the same particle shown in panel E displays a random then constrained diffusion.Taken together, our findings demonstrate that the movement of the pVLPs is driven by actin. Analysis of the pVLPs trajectories also suggests that the motion of pVLPs on actin enables further addition of VP40 molecules. These findings raise important questions regarding contemporary understanding of Ebola assembly and egress. VP40 lacks a consensus actin-binding motif, suggesting an adaptor protein such as an actin motor protein may function in this process. For instance, Myo10 has been found to be essential to Marburg virus release (6); however, Marburg VP40-Myo10 direct interactions were not observed, suggesting other cellular adaptor proteins may function in this process. Given the pathogenic nature of the Ebola virus and the necessity of VP40 to the assembly and egress of the virus (8), the VP40-actin coordination represents, to us, a novel target for therapeutic development.     

Number and brightness analysis of LRRK2 oligomerization in live cells

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.