Measurement of percentage dose at the surface for a 6 MV photon beam

AimTo evaluate if a radiochromic film (RF) Gafchromic EBT3 is suitable for surface dose measurements of radiotherapy treatments performed with a 6 MV linear accelerator. Two aspects of RF were analyzed, beam energy dependence and surface dose determination.BackgroundThe measurements done at the surface or near the radiation source are done without charged electronic equilibrium and also have contribution of electron contamination. The detectors used for these measurements should not alter the dose to the target. To counteract these dosimetric problems it is proposed to do the measurements with radiochromic films which are thin detectors and have tissue equivalent properties.Materials and MethodsThe measurements were done using a Novalis linear accelerator (LINAC) with nominal energy of 6 MV. To determine the surface dose, the total scatter factors (TSF) of three different field sizes were measured in a water phantom at 5 cm depth. Energy dependence of EBT3 was studied at three different depths, using a solid water phantom. The surface measurements were done with the RF for the same field sizes of the TSF measurements. The value of the percentage depth dose was calculated normalizing the doses measured in the RF with the LINAC output, at 5 cm depth, and the TSF.ResultsThe radiochromic films showed almost energy independence, the differences between the curves are 1.7% and 1.8% for the 1.5 cm and 10 cm depth, respectively. The percentage depth doses values at the surface measured for the 10 cm × 10 cm, 5 cm × 5 cm and 1 cm × 1 cm were 26.1 ± 1.3%, 21.3 ± 2.4% and 20.2 ± 2.6%, respectively.ConclusionsThe RF-EBT3 seems to be a detector suitable for measurements of the dose at the surface. This suggests that RF-EBT3 films might be good candidates as detectors for in vivo dosimetry.     

Autophagy and neuropeptides at the crossroad for parasites: To survive or to die?

We have recently demonstrated that some neuropeptides act as potent endogenous antiparasitic factors. These molecules kill trypanosomes through complex mechanisms that are difficult to escape by the parasite. Neuropeptides are endocytosed by the parasite, disrupt lysosome integrity, and alter the cellular compartmentalization of glycolytic enzymes. This promotes an energetic metabolism failure that initiates an autophagic-like cell death. The concept of autophagy is new for parasites and was mainly associated with differentiation or stress events. Here, we propose that this form of programmed cell death probably co-evolved with parasite-induced-neuropeptides after host infection, as a survival strategy favoring parasite transmission for a longer time by keeping the host alive.     

Colocalization of the genes coding for the α3 and β3 subunits of soluble guanylyl cyclase to human chromosome 4 at q31.3–q33

We have determined the chromosomal location of the human genes coding for the ₃ and ₃ subunits of soluble guanylyl cyclase (GC-S). The study was performed by in situ hybridization of human metaphase spreads with two human cDNA probes, containing the coding sequences of the GC-S ₃ and ₃ subunits, respectively. Each probe gave a strong specific signal on chromosome 4 at the 4q31.3–4q33 region, with the maximal signal in the 4q32 band. The colocalization of both genes in 4q32 represents an interesting feature for the coordinated regulation of expression of both GC-S subunits.     

In-vivo dosimetry for field sizes down to 6 × 6 mm2 in shaped beam radiosurgery with microMOSFET

The aim of this study is to evaluate microMOSFET as in-vivo dosimeter in 6 MV shaped-beam radiosurgery for field sizes down to 6 × 6 mm². A homemade build-up cap was developed and its use with microMOSFET was evaluated down to 6 × 6 mm². The study with the homemade build-up cap was performed considering its influence on field size over-cover occurring at surface, achievement of the overall process of electronic equilibrium, dose deposition along beam axis and dose attenuation. An optimized calibration method has been validated using MOSFET in shaped-beam radiosurgery for field sizes from 98 × 98 down to 18 × 18 mm². The method was detailed in a previous study and validated in irregular field shapes series measurements performed on a head phantom. The optimized calibration method was applied to microMOSFET equipped with homemade build-up cap down to 6 × 6 mm². Using the same irregular field shapes, dose measurements were performed on head phantom. MicroMOSFET results were compared to previous MOSFET ones. Additional irregular field shapes down to 8.8 × 8.8 mm² were studied with microMOSFET. Isocenter dose attenuation due to the homemade build-up cap over the microMOSFET was near 2% irrespective of field size. Our results suggested that microMOSFET equipped with homemade build-up cap is suitable for in-vivo dosimetry in shaped-beam radiosurgery for field sizes down to 6 × 6 mm² and therefore that the required build-up cap dimensions to perform entrance in-vivo dosimetry in small-fields have to ensure only partial charge particle equilibrium.     

mTOR and cancer: reason for dancing at the crossroads?

Recent successes using Gleevec for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors have provided proof that strategies to target signal transduction pathways mutated in human cancers can work. However, the application of this strategy to other cancers has been slow. Central to alleviating this impedance is the molecular characterization of the tumors. There is an urgent need to translate basic scientific findings into relevant, clinically applicable molecular diagnostic assays.     

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.     

Measurements of urinary leptin and capillary leptin: alternative tools for the assessment of the leptin status?

OBJECTIVES: The aim of this study was to check whether leptin is reliably measurable in urine samples of children, adolescents, and adults and to examine whether capillary leptin measurements can be utilized as an alternative tool to assess the leptin status. METHODS: Two studies were performed. In both studies, leptin was quantified by an ultrasensitive and highly specific enzyme immunoassay (ELISA; R & D Systems). Anthropometric measures were taken from all study subjects, and body fat was calculated using skinfold thickness measurements. In study 1, leptin was analyzed in 24-hour urine samples of 155 healthy children and adolescents and 26 healthy adults after a methodological modification of the assay necessary for urine analysis. In study 2, venous and capillary blood samples were collected in 26 healthy adults within 10 min on the same day. RESULTS: After adapting the assay system to urine matrix, the detection range was 20-160 pg/ml. Only in 2 of 181 urine samples reproducibly measurable urinary leptin concentrations in the lowest detection range were found. In study 2, a close correlation was found between log capillary and log venous leptin concentrations (r = 0.98, p < 0.001) and between log capillary as well as log venous leptin levels and percent body fat (r = 0.86, p < 0.001). CONCLUSIONS: Our results based on one of the most specific and sensitive ELISAs currently available show that leptin is generally undetectable in the urine from healthy children, adolescents and adults. Thus, urinary leptin excretion cannot be used as a noninvasive marker of the leptin status. Our findings in healthy adults show that the merely moderately invasive determination of capillary leptin allows a reliable assessment of the individual leptin status and may be used instead of venous leptin as a biochemical indicator of body fatness.     

Room at the top? Minority mobility and the transition to demographic diversity in the USA

We examine the ramifications of the demographic transition to diversity in the USA through an analysis of changes at the top of the occupational hierarchy, as glimpsed in recent census data. We find evidence that, among the incumbents of better-paid occupations, the percentage of non-Hispanic whites, the historically dominant majority, is sharply declining, while the proportion of immigrant-origin minorities, Asians, both foreign and US born, and US-born Hispanics, is growing. African Americans, or US-born blacks, are not so far sharing much in this growth. However, whites still retain important advantages in terms of occupational placement net of education and in terms of earnings net of occupational placement. We conclude overall that demographic shifts are highly likely to drive further diversification at the top of the US workforce; the question of whether whites can hold on to their advantages in the face of these changes cannot be answered yet.     

Is functional hypertrophy and specific force coupled with the addition of myonuclei at the single muscle fiber level?

Muscle force is typically proportional to muscle size, resulting in constant force normalized to muscle fiber cross-sectional area (specific force). Mice overexpressing insulin-like growth factor-1 (IGF-1) exhibit a proportional gain in muscle force and size, but not the myostatin-deficient mice. In an attempt to explore the role of the cytoplasmic volume supported by individual myonuclei [myonuclear domain (MND) size] on functional capacity of skeletal muscle, we have investigated specific force in relation to MND and the content of the molecular motor protein, myosin, at the single muscle fiber level from myostatin-knockout (Mstn(-/-)) and IGF-1-overexpressing (mIgf1(+/+)) mice. We hypothesize that the addition of extra myonuclei is a prerequisite for maintenance of specific force during muscle hypertrophy. A novel algorithm was used to measure individual MNDs in 3 dimensions along the length of single muscle fibers from the fast-twitch extensor digitorum longus and the slow-twitch soleus muscle. A significant effect of the size of individual MNDs in hypertrophic muscle fibers on both specific force and myosin content was observed. This effect was muscle cell type specific and suggested there is a critical volume individual myonuclei can support efficiently. The large MNDs found in fast muscles of Mstn(-/-) mice were correlated with the decrement in specific force and myosin content in Mstn(-/-) muscles. Thus, myostatin inhibition may not be able to maintain the appropriate MND for optimal function.     

NTPase and 5′ to 3′ RNA Duplex-Unwinding Activities of the Hepatitis E Virus Helicase Domain

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg²⁺ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m⁷G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV.     

CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly

Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.     

Recombination hot spots within the I region of the mouse H-2 complex map to the E β and E α genes

Recombinant mouse strains with crossovers in the I region of the H-2 major histocompatibility complex were examined by restriction fragment analysis for the presence of polymorphic restriction sites within the E and E genes. Nine recombinant mouse strains were shown to have crossed over within a 5 kb DNA segment that contains the large intron between the second and third exons of the E gene. These results are in accord with previous studies mapping a recombination hot spot within this gene. Seven recombinant mouse strains between the p and k haplotypes were shown to have crossed over in a 6 kb segment within the E gene. These results show the existence of a recombination hot spot within the E gene. Comparison of the H-2 haplotypes involved in these two recombination hot spots suggests that a specific DNA sequence in b, s, f, and q haplotypes may act to promote recombination in the E gene and a specific DNA sequence in the p haplotype may act to promote recombination in the E gene.     

14-3-3 protein and ATRAP bind to the soluble class IIB phosphatidylinositol transfer protein RdgBβ at distinct sites

PITPs (phosphatidylinositol transfer proteins) are characterized by the presence of the PITP domain whose biochemical properties of binding and transferring PI (phosphatidylinositol) are well studied. Despite their wide-spread expression in both unicellular and multicellular organisms, they remain functionally uncharacterized. An emerging theme is that individual PITPs play highly specific roles in either membrane trafficking or signal transduction. To identify specific roles for PITPs, identification of interacting molecules would shed light on their molecular function. In the present paper, we describe binding partners for the class IIB PITP RdgBβ (retinal degeneration type?Bβ). RdgBβ is a soluble PITP but is unique in that it contains a region of disorder at its C-terminus following its defining N-terminal PITP domain. The C-terminus of RdgBβ is phosphorylated at two serine residues, Ser274 and Ser299, which form a docking site for 14-3-3 proteins. Binding to 14-3-3 proteins protects RdgBβ from degradation that occurs at the proteasome after ubiquitination. In addition to binding 14-3-3, the PITP domain of RdgBβ interacts with the Ang II (angiotensin II)-associated protein ATRAP (Ang II receptor-associated protein). ATRAP is also an interacting partner for the AT1R (Ang II type?1 receptor). We present a model whereby RdgBβ functions by being recruited to the membrane by ATRAP and release of 14-3-3 from the C-terminus allows the disordered region to bind a second membrane to create a membrane bridge for lipid transfer, possibly under the control of Ang II.