Comprehensive radiation and imaging isocenter verification using NIPAM kV-CBCT dosimetry.

PURPOSE: To develop and demonstrate a comprehensive method to directly measure radiation isocenter uncertainty and coincidence with the cone-beam computed tomography (kV-CBCT) imaging coordinate system that can be carried out within a typical quality assurance (QA) time slot. METHODS: An N-isopropylacrylamide (NIPAM) three-dimensional (3D) dosimeter for which dose is observed as increased electron density in kV-CBCT is irradiated at eight couch/gantry combinations which enter the dosimeter at unique orientations. One to three CBCTs are immediately acquired, radiation profile is detected per beam, and displacement from imaging isocenter is quantified. We performed this test using a 5 mm diameter MLC field, and 7.5 and 4 mm diameter cones, delivering approximately 16 Gy per beam. CBCT settings were 1035-4050 mAs, 80-125 kVs, smooth filter, 1 mm slice thickness. The two-dimensional (2D) displacement of each beam from the imaging isocenter was measured within the planning system, and Matlab code developed in house was used to quantify relevant parameters based on the actual beam geometry. Detectability of the dose profile in the CBCT was quantified as the contrast-to-noise ratio (CNR) of the irradiated high-dose regions relative to the surrounding background signal. Our results were compared to results determined by the traditional Winston-Lutz test, film-based "star shots," and the vendor provided machine performance check (MPC). The ability to detect alignment errors was demonstrated by repeating the test after applying a 0.5 mm shift to the MLCs in the direction of leaf travel. In addition to radiation isocenter and coincidence with CBCT origin, the analysis also calculated the actual gantry and couch angles per beam. RESULTS: Setup, MV irradiation, and CBCT readout were carried out within 38 min. After subtracting the background signal from the pre-CBCT, the CNR of the dosimeter signal from the irradiation with the MLCs (125 kVp, 1035 mAs, n = 3), 7.5 mm cone (125 kVp, 1035 mAs, n = 3), and 4 mm cone (80 kVp, 4050 mAs, n = 1) was 5.4, 5.9, and 2.9, respectively. The minimum radius that encompassed all beams calculated using the automated analysis was 0.38, 0.48, and 0.44 mm for the MLCs, 7.5 mm cone, and 4 mm cone, respectively. When determined manually, these values were slightly decreased at 0.28, 0.41, and 0.40 mm. For comparison, traditional Winston-Lutz test with MLCs and MPC measured the 3D isocenter radius to be 0.24 mm. Lastly, when a 0.5 mm shift to the MLCs was applied, the smallest radius that intersected all beams increased from 0.38 to 0.90 mm. The mean difference from expected value for gantry angle was 0.19 ± 0.29°, 0.17 ± 0.23°, and 0.12 ± 0.14° for the MLCs, 7.5 mm cone, and 4 mm cone, respectively. The mean difference from expected for couch angle was -0.07 ± 0.28°, -0.08 ± 0.66°, and 0.04 ± 0.25°. CONCLUSIONS: This work demonstrated the feasibility of a comprehensive isocenter verification using a NIPAM dosimeter with sub-mm accuracy which incorporates evaluation of coincidence with imaging coordinate system, and may be applicable to all SRS cones as well as MLCs.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292-296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.

Removal of the cardiac myosin regulatory light chain increases isometric force production.

The myosin neck, which is supported by the interactions between light chains and the underlying alpha-helical heavy chain, is thought to act as a lever arm to amplify movements originating in the globular motor domain. Here, we studied the role of the cardiac myosin regulatory light chains (RLCs) in the capacity of myosin to produce force using a novel optical-trap-based isometric force in vitro motility assay. We measured the isometric force and actin filament velocity for native porcine cardiac (PC) myosin, RLC-depleted PC (PC(depl)) myosin, and PC myosin reconstituted with recombinant bacterially expressed human cardiac RLC (PC(recon)). RLC depletion reduced unloaded actin filament velocity by 58% and enhanced the myosin-based isometric force approximately 2-fold. No significant change between PC and PC(depl) preparations was observed in the maximal rate of actin-activated myosin ATPase activity. Reconstitution of PC(depl) myosin with human RLC partially restored the velocity and force levels to near untreated values. The reduction in unloaded velocity after RLC extraction is consistent with the myosin neck acting as a lever, while the enhancement in isometric force can be directly related to enhancement of unitary force. The force data are consistent with a model in which the neck region behaves as a cantilevered beam.

Prevalence of undernutrition among children in the Garhwal Himalayas.

BACKGROUND: Nutritional status is the best indicator of the global well-being of children. In India every third child is underweight, whereas in the Garhwal Himalayas only one-fifth of the children are nutritionally normal. Information regarding the causes of such low nutritional status in children of Garhwal is lacking. OBJECTIVE: The present study was designed to identify factors that may influence the nutritional status of the children of the Garhwal Himalayas. Stunting, signifying long-term irreversible chronic undernutrition, was studied, along with wasting, which signifies acute undernutrition among children. METHODS: Forty families from one town and one village in each of the three major agroclimatic situations (high, middle, and low hills) were selected at random (total 240 families). The nutritional status of 353 children (0 to 12 years of age) was assessed by nutritional anthropometry and compared with tables of weight-for-age and height-for-age z-scores (WAZ and HAZ) identifying wasting and stunting, respectively, and compared with the World Health Organization (WHO) Growth Reference charts 2007. Chi-square test was applied to analyze the effects of age, sex, altitude, and area on the nutritional status of children. RESULTS: Of the 353 children studied, 38.2% belonged to the school-aged group, with 7% more girls than boys. Only 11.3% of the children had normal HAZ while 10.3% had normal WAZ. The largest percentages of children (31.8% HAZ and 29.1% WAZ) were clustered in the SD1 group, that is the severely stunted or wasted group. In every age group except the 9- to 12-year group, most of the children were found in the WAZ SD1 category. Gender appeared to influence the SD1 category significantly. There were 17.0% severely stunted girls (SD1), as compared to 14.8% boys; while the percentages of WAZ girls was 16.1% and boys was 13%. The situation in the urban areas was slightly better: 6.4% of the sample had normal HAZ and 7.1% had normal WAZ, compared with 4.4% and 4.6% of the rural children. In all three agroclimatic situations, the largest percentage of children fell in the HAZ SD1 (stunted) category (15.3% in the high hills, 7.3% in the middle hills, and 9.5% in the low hills). There is a significant effect of area (rural vs. urban) on stunting and wasting among children. The child's sex affects stunting only. The other two variables, altitude and age, do not show a significant relationship with stunting or wasting. CONCLUSIONS: The rate of malnutrition among children in the Garhwal is very high. The majority are severely stunted and wasted, indicating a high prevalence of both chronic and acute malnutrition. Only the area of residence (rural or urban) had an influence on a child's nutritional status.

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY.

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction.

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.

First passage time of N excluded-volume particles on a line.

Motivated by recent single-molecule studies of proteins sliding on a DNA molecule, we explore the targeting dynamics of particles ("proteins") sliding diffusively along a line ("DNA") in search of their target site (specific target sequence). At lower particle densities, one observes an expected reduction of the mean first passage time proportional to N(-2), with corrections at higher concentrations. We explicitly take adsorption and desorption effects, to and from the DNA, into account. For this general case, we also consider finite-size effects when the continuum approximation based on the number density of particles breaks down. Moreover, we address the first-passage-time problem of a tagged particle diffusing among other particles.

Theory of electrostatically regulated binding of T4 gene 32 protein to single- and double-stranded DNA.

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA binding protein, which is essential for DNA replication, recombination, and repair. In a recent article, we described a new method using single DNA molecule stretching measurements to determine the noncooperative association constants K(ds) to double-stranded DNA for gp32 and *I, a truncated form of gp32. In addition, we developed a single molecule method for measuring K(ss), the association constant of these proteins to single-stranded DNA. We found that in low salt both K(ds) and K(ss) have a very weak salt dependence for gp32, whereas for *I the salt dependence remains strong. In this article we propose a model that explains the salt dependence of gp32 and *I binding to single-stranded nucleic acids. The main feature of this model is the strongly salt-dependent removal of the C-terminal domain of gp32 from its nucleic acid binding site that is in pre-equilibrium to protein binding to both double-stranded and single-stranded nucleic acid. We hypothesize that unbinding of the C-terminal domain is associated with counterion condensation of sodium ions onto this part of gp32, which compensates for sodium ion release from the nucleic acid upon its binding to the protein. This results in the salt-independence of gp32 binding to DNA in low salt. The predictions of our model quantitatively describe the large body of thermodynamic and kinetic data from bulk and single molecule experiments on gp32 and *I binding to single-stranded nucleic acids.

Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements.

Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K(ds)) to double-stranded DNA to determine K(ds) as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K(ss), the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD.

Target search of N sliding proteins on a DNA.

At low to moderate ambient salt concentrations, DNA-binding proteins bind relatively tightly to DNA, and only very rarely detach. Intersegmental transfer due to DNA-looping can be excluded by applying an external pulling force to the DNA molecule. Under such conditions, we explore the targeting dynamics of N proteins sliding diffusively along DNA in search of their specific target sequence. At lower densities of binding proteins, we find a reduction of the characteristic search time proportional to N(-2), with corrections at higher concentrations. Rates for detachment and attachment of binding proteins are incorporated in the model. Our findings are in agreement with recent single molecule studies in the presence of bacteriophage T4 gene 32 protein for which the unbinding rate is much lower than the specific binding rate.

Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein.

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair. While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA). However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA. In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein. This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA. The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit. We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess. Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I. Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.

Kinetic regulation of single DNA molecule denaturation by T4 gene 32 protein structural domains.

Bacteriophage T4 gene 32 protein (gp32) specifically binds single-stranded DNA, a property essential for its role in DNA replication, recombination, and repair. Although on a thermodynamic basis, single-stranded DNA binding proteins should lower the thermal melting temperature of double-stranded DNA (dsDNA), gp32 does not. Using single molecule force spectroscopy, we show for the first time that gp32 is capable of slowly destabilizing natural dsDNA. Direct measurements of single DNA molecule denaturation and renaturation kinetics in the presence of gp32 and its proteolytic fragments reveal three types of kinetic behavior, attributable to specific protein structural domains, which regulate gp32's helix-destabilizing capabilities. Whereas the full-length protein exhibits very slow denaturation kinetics, a truncate lacking the acidic C-domain exhibits much faster kinetics. This may reflect a steric blockage of the DNA binding site and/or a conformational change associated with this domain. Additional removal of the N-domain, which is needed for binding cooperativity, further increases the DNA denaturation rate, suggesting that both of these domains are critical to the regulation of gp32's helix-destabilization capabilities. This regulation is potentially biologically significant because uncontrolled helix-destabilization would be lethal to the cell. We also obtain equilibrium measurements of the helix-coil transition free energy in the presence of these proteins for the first time.