Extraction method combining saponin and trehalose useful for analyzing fragile intermolecular association.

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are well-established methodologies to analyze protein expression and intermolecular interaction. Composition of extraction and washing buffer for preparing protein is important to accomplish experimental purpose. Various kinds of detergents are included in buffer to adjust extraction efficiency and washing effect. Among them, Triton X-100 (Tx-100), Nonidet P-40 (NP40), deoxycholic acid (DOC) and SDS are generally used according to experimental purpose and characteristic features of protein of interest. In some cases, general detergents disrupt intermolecular interaction and make it impossible to analyze molecular relation of protein of interest with its binding partners. In this study, we propose saponin, a natural detergent, is useful for co-immunoprecipitation when analyzing fragile intermolecular interactions, in which dystrophin and dystroglycan are used as a representative interaction. One of the most notable findings in this report is that intermolecular association between dystrophin and dystroglycan is maintained in saponin buffer whereas general detergents, such as Tx-100, NP40 and DOC, dissociate its binding. Furthermore, supplementation of trehalose, which has been shown to act as a molecular chaperone, facilitates efficient detection of dystrophin-dystroglycan macromolecular complex in co-IP assay. Importantly, the extraction buffer comprising 3 % saponin, 0.5 M trehalose and 0.05 % Tx-100 (we named it STX buffer) is applicable to co-IP for another molecular interaction, N-cadherin and ß-catenin, indicating that this methodology can be used for versatile proteins of interest. Thus, STX buffer emerges as an alternative extraction method useful for analyzing fragile intermolecular associations and provides opportunity to identify complex interactomes, which may facilitate proteome-research and functional analysis of proteins of interest.

Pkd2, mutations linking to autosomal dominant polycystic kidney disease, localizes to the endoplasmic reticulum and regulates calcium signaling in fission yeast.

Autosomal dominant polycystic kidney disease (ADPKD) is a renal disorder caused by mutations in the PKD2 gene, which encodes polycystin-2/Pkd2, a transient receptor potential channel. The precise role of Pkd2 in cyst formation remains unclear. The fission yeast Schizosaccharomyces pombe has a putative transient receptor potential channel, Pkd2, which shares similarities with human Pkd2. In this study, truncation analyses of fission yeast Pkd2 were conducted to investigate its localization and function. The results revealed that Pkd2 localizes not only to the plasma membrane but also to the endoplasmic reticulum (ER) in fission yeast. Furthermore, Pkd2 regulates calcium signaling in fission yeast, with the transmembrane domains of Pkd2 being sufficient for these processes. Specifically, the C-terminal region of Pkd2 plays a crucial role in the regulation of calcium signaling. Interestingly, human Pkd2 also localized to the ER and had some impact on calcium signaling in fission yeast. However, human Pkd2 failed to suppress the loss of fission yeast Pkd2. These findings indicate that hPkd2 may not completely substitute for cellular physiology of fission yeast Pkd2. This study provides insights into the localization and functional characteristics of Pkd2 in fission yeast, contributing to our understanding of the pathogenesis of ADPKD.

Leukemia inhibitory factor shortens primary cilia by upregulating C-C motif chemokine 2 in human neural stem/progenitor cells.

Primary cilia on neural stem/progenitor cells (NSPCs) play an important role in determining cell fate, although the regulatory mechanisms involved in the ciliogenesis remain largely unknown. In this study, we analyzed the effect of the leukemia inhibitory factor (LIF) for the primary cilia in immortalized human NSPCs. LIF withdrawal elongated the primary cilia length, whereas the addition of LIF shortened it. Microarray gene expression analysis revealed that differentially expressed genes (DEGs) associated with LIF treatment were related with the multiple cytokine signaling pathways. Among the DEGs, C-C motif chemokine 2 (CCL2) had the highest ranking and its increase in the protein concentration in the NSPCs-conditioned medium after the LIF treatment was confirmed by ELISA. Interestingly, we found that CCL2 was a negative regulator of cilium length, and LIF-induced shortening of primary cilia was antagonized by CCL2-specific antibody, suggesting that LIF could influence cilia length via upregulating CCL2. The shortening effect of LIF and CCL2 on primary cilia was also observed in SH-SY5Y cells. The results of the study suggested that the LIF-CCL2 axis may well be a regulator of NSPCs and its primary cilia length, which could affect multiple cellular processes, including NSPC proliferation and differentiation.

Influence of dose calculation algorithms on the helical diode array using volumetric-modulated arc therapy for small targets.

BACKGROUND: For patient-specific quality assurance (PSQA) for small targets, the dose resolution can change depending on the characteristics of the dose calculation algorithms. PURPOSE: This study aimed to evaluate the influence of the dose calculation algorithms Acuros XB (AXB), anisotropic analytical algorithm (AAA), photon Monte Carlo (pMC), and collapsed cone (CC) on a helical diode array using volumetric-modulated arc therapy (VMAT) for small targets. MATERIALS AND METHODS: ArcCHECK detectors were inserted with a physical depth of 2.9 cm from the surface. To evaluate the influence of the dose calculation algorithms for small targets, rectangular fields of 2×100, 5×100, 10×100, 20×100, 50×100, and 100×100 mm2 were irradiated and measured using ArcCHECK with TrueBeam STx. A total of 20 VMAT plans for small targets, including the clinical sites of 19 brain metastases and one spine, were also evaluated. The gamma passing rates (GPRs) were evaluated for the rectangular fields and the 20 VMAT plans using AXB, AAA, pMC, and CC. RESULTS: For rectangular fields of 2×100 and 5×100 mm2, the GPR at 3%/2 mm of AXB was < 50% because AXB resulted in a coarser dose resolution with narrow beams. For field sizes > 10×100 mm2, the GPR at 3%/2 mm was > 88.1% and comparable for all dose calculation algorithms. For the 20 VMAT plans, the GPRs at 3%/2 mm were 79.1 ± 15.7%, 93.2 ± 5.8%, 94.9 ± 4.1%, and 94.5 ± 4.1% for AXB, AAA, pMC, and CC, respectively. CONCLUSION: The behavior of the dose distribution on the helical diode array differed depending on the dose calculation algorithm for small targets. Measurements using ArcCHECK for VMAT with small targets can have lower GPRs owing to the coarse dose resolution of AXB around the detector area.

Generation of dystrophin short product-specific tag-insertion mouse: distinct Dp71 glycoprotein complexes at inhibitory postsynapse and glia limitans.

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA-tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71, although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinß (Dtnb), whereas glia-associated Dp71 did with Dg and dystrobrevinα (Dtna). Thus, our data indicate that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

Dystroglycan regulates proper expression, submembranous localization and subsequent phosphorylation of Dp71 through physical interaction.

Dystrophin-dystroglycan complex (DGC) plays important roles for structural integrity and cell signaling, and its defects cause progressive muscular degeneration and intellectual disability. Dystrophin short product, Dp71, is abundantly expressed in multiple tissues other than muscle and is suspected of contributing to cognitive functions; however, its molecular characteristics and relation to dystroglycan (DG) remain unknown. Here, we report that DG physically interacts with Dp71 in cultured cells. Intriguingly, DG expression positively and DG knockdown negatively affected the steady-state expression, submembranous localization and subsequent phosphorylation of Dp71. Mechanistically, two EF-hand regions along with a ZZ motif of Dp71 mediate its association with the transmembrane proximal region, amino acid residues 788-806, of DG cytoplasmic domain. Most importantly, the pathogenic point mutations of Dp71, C272Y in the ZZ motif or L170del in the second EF-hand region, impaired its binding to DG, submembranous localization and phosphorylation of Dp71, indicating the relevance of DG-dependent Dp71 regulatory mechanism to pathophysiological conditions. Since Dp140, another dystrophin product, was also regulated by DG in the same manner as Dp71, our results uncovered a tight molecular relation between DG and dystrophin, which has broad implications for understanding the DGC-related cellular physiology and pathophysiology.

Single-cell transcriptome analysis of fractional CO2 laser efficiency in treating a mouse model of alopecia.

OBJECTIVES: Hair loss, including alopecia, is a common dermatological issue worldwide. At present, the application of fractional carbon dioxide (CO2 ) laser in the treatment of alopecia has been documented; however, the results vary between reports. These varying results may be due to the limited knowledge of cellular action in laser-irradiated skin. The objective of this study was to investigate the molecular and cellular mechanisms of laser treatment under effective conditions for hair cycle initiation. METHODS: A fractional CO2 laser was applied and optimized to initiate the hair cycle in a mouse model of alopecia. Several cellular markers were analyzed in the irradiated skin using immunofluorescence staining. Cellular populations and their comprehensive gene expression were analyzed using single-cell RNA sequencing and bioinformatics. RESULTS: The effective irradiation condition for initiating the hair cycle was found to be 15 mJ energy/spot, which generates approximately 500 µm depth columns, but does not penetrate the dermis, only reaching approximately 1 spot/mm2 . The proportion of macrophage clusters significantly increased upon irradiation, whereas the proportion of fibroblast clusters decreased. The macrophages strongly expressed C-C chemokine receptor type 2 (Ccr2), which is known to be a key signal for injury-induced hair growth. CONCLUSIONS: We found that fractional CO2 laser irradiation recruited Ccr2 positive macrophages, and induced hair regrowth in a mouse alopecia model. These findings may contribute to the development of stable and effective fractional laser irradiation conditions for human alopecia treatment.

Variation in target volume and centroid position due to breath holding during four-dimensional computed tomography scanning: A phantom study.

This study investigated the effects of respiratory motion, including unwanted breath holding, on the target volume and centroid position on four-dimensional computed tomography (4DCT) imaging. Cine 4DCT images were reconstructed based on a time-based sorting algorithm, and helical 4DCT images were reconstructed based on both the time-based sorting algorithm and an amplitude-based sorting algorithm. A spherical object 20 mm in diameter was moved according to several simulated respiratory motions, with a motion period of 4.0 s and maximum amplitude of 5 mm. The object was extracted automatically, and the target volume and centroid position in the craniocaudal direction were measured using a treatment planning system. When the respiratory motion included unwanted breath-holding times shorter than the breathing cycle, the root mean square errors (RSME) between the reference and imaged target volumes were 18.8%, 14.0%, and 5.5% in time-based images in cine mode, time-based images in helical mode, and amplitude-based images in helical mode, respectively. In helical mode, the RSME between the reference and imaged centroid position was reduced from 1.42 to 0.50 mm by changing the reconstruction method from time- to amplitude-based sorting. When the respiratory motion included unwanted breath-holding times equal to the breathing cycle, the RSME between the reference and imaged target volumes were 19.1%, 24.3%, and 15.6% in time-based images in cine mode, time-based images in helical mode, and amplitude-based images in helical mode, respectively. In helical mode, the RSME between the reference and imaged centroid position was reduced from 1.61 to 0.83 mm by changing the reconstruction method from time- to amplitude-based sorting. With respiratory motion including breath holding of shorter duration than the breathing cycle, the accuracies of the target volume and centroid position were improved by amplitude-based sorting, particularly in helical 4DCT.

Functional assessment of improvement of myocardial ischemia using coronary flow velocity reserve after coronary artery bypass surgery in hemodialysis.

BACKGROUND: In patients with end-stage renal disease requiring hemodialysis (HD patients), myocardial ischemia after coronary artery disease is a major cause of mortality. Coronary pathophysiology, namely myocardial microvascular dysfunction, appears to differ from patients not requiring HD (non-HD patients). OBJECTIVES: We compared functional improvement of myocardial ischemia after coronary artery bypass surgery (CABG) between HD and non-HD patients by transthoracic coronary flow velocity reserve (CFVR). METHODS: We retrospectively reviewed isolated CABG patients from between 2008 and 2017. Finally, 161 patients were enrolled; each underwent pre- and postoperative CFVR assessment, and left anterior descending (LAD) artery revascularization with "in-situ" internal mammary artery (IMA). Graft patency was confirmed, and after successful CABG, postoperative CFVR improvement between the two groups was compared. RESULTS: Preoperative CFVR value in group H was 1.81 ± 0.52, group N was 1.93 ± 0.66. There was no significant difference between the groups. IMA to LAD grafts were patent in postoperative evaluation in all patients. Postoperative CFVR in group H was 2.48 ± 0.72 and group N was 2.83 ± 0.73 (P = .042). Significant difference was observed. CONCLUSION: In both groups, CFVR values improved after successful CABG, but postoperative CFVR values were significant different. In younger populations CFVR values are generally higher. Our HD group was significantly younger than the non-HD group, but CFVR values were postoperatively significantly lower. CFVR values are reportedly affected by both epicardial and microcoronary circulation. In this study population, as all grafts to the LAD were patent, the lower CFVR value in the HD group was considered to have resulted in microvascular disorders.

[Comparison of Monitor Unit between Electron Monte Carlo Algorithm by Different Dose Normalization Methods and Conventional Manual Calculation].

The purpose of this study was to evaluate the discrepancy between the monitor unit (MU) calculated by different dose normalization methods in the electron Monte Carlo (eMC) algorithm and the conventional manual MU. In the water phantom condition, the manual MU obtained from the measured output factor was compared with the calculated MU by the eMC algorithm, using 24 different irradiation field shapes and several different energies of electron beam. In the breast boost condition, calculated MUs by both calculation methods were evaluated for 45 cases. As a result, the MUs computed by the eMC algorithm in the water phantom varied according to the dose normalization methods, and the mean±standard deviation of the difference between the manual and calculated MU were 1.1±1.4%, 0.0±1.0% and 0.4±1.2% in peak depth normalization (PN), no plan normalization (NPN) and 100% at body maximum (100%BM), respectively. In breast-boost cases, the MU difference between the manual and the calculated MU were 6.1±3.7%, 3.4±2.8% and 1.1±2.9% in PN, NPN and 100%BM, respectively. We revealed that the resultant MU calculated by eMC algorithm was dependent on the dose normalization method and the averaged differences exceeded 6% in PN, especially in breast boost condition. When using the eMC in the breast boost condition, it is desirable to select an appropriate dose normalization method according to dose prescription policies at each facility.

Dp71 is regulated by phosphorylation and ubiquitin-proteasome system in neuronal cells.

The Dystrophin (Dp) gene is responsible for Duchenne muscular dystrophy (DMD), which is characterized by progressive muscular degeneration and variable degrees of cognitive impairment. Although Dp71 is the most abundant among the Dp isoforms in the brain, the regulatory mechanisms of the related expression levels have not been elucidated. In this study, we found that the constitutive expression levels of Dp71 in PC12 cells were sensitive to proteasomal inhibition. The ectopic expression of FLAG-tagged ubiquitin revealed that Dp71 was ubiquitinated intracellularly. Interestingly, proteasomal inhibition was accompanied by a posttranslational accumulation of modified Dp71, which was restored by protein phosphatase treatment in vitro, indicating that phosphorylation is responsible for the modification and affects the proteasome-dependent degradation of Dp71. Proteasomal activity-sensitive phosphorylated Dp71 is closely associated with syntrophin, a well-known binding partner of Dp71, and syntrophin is also regulated by proteasomal activity in a similar way to Dp71, suggesting that the posttranslational regulatory machinery for Dp71 level is coupled with Dp71-syntrophin molecular complex. Taken together, our results indicated that the expression levels of Dp71 are posttranslationally regulated by the phosphorylation-ubiquitin-proteasomal pathway, which may indicate the presence of regulatory mechanisms underlying the proteostasis of both Dp and its molecular complex, which may lead to better therapeutic approaches for the treatment of Dp-related diseases.

Transcutaneous drug delivery by liposomes using fractional laser technology.

OBJECTIVE: Transdermal delivery of hydrophilic peptides remains a challenge due to their poor cellular uptake and transdermal penetration. We hypothesize that combination of a CO2 fractional laser to enhance percutaneous absorption and liposomes as transdermal carriers would improve skin penetration of hydrophilic drugs. STUDY DESIGN: NA. METHODS: Liposomes were prepared using membrane fusion lipid dioleoylphosphatidylethanolamine, and used to deliver 5-carboxyfluorescein (CF) and fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC) as model hydrophilic peptide drugs. Liposome size was estimated by dynamic light scattering. Liposome uptake into murine macrophage cells and penetration or permeation into Yucatan micropig skin after irradiation by CO2 fractional laser at varying energy levels (laser power and exposure duration) were investigated using Franz cell and fluorescence microscopy. Oxidative damage to the irradiated mouse skin was assessed by electron spin resonance. RESULTS: Size of CF and OVA-FITC encapsulated liposomes was 324 ± 75 nm. Cellular uptake of OVA-FITC delivered by liposomes was 10-fold higher (1,370 relative fluorescence units, RFU) than delivered in solution form (130 RFU). Fractional laser irradiation increased skin permeation rate of CF liposomes (0-10%) and OVA-FITC liposomes (4-40%) in a dose-dependent manner. Although peeling off the stratum corneum facilitated CF liposome penetration at low energy levels (2.69-3.29 J/cm2 ; 10-20 W for 500 µs), drug permeation was similar (7-8%) in peeled or untreated skin at higher laser energy levels (6.06 J/cm2 ; 20 W for 1,500 µs). FITC penetrated deeper in the skin after laser irradiation. However, OH, O2-, and VC reactive oxygen species were generated upon irradiation of the skin with a fractional CO2 laser. CONCLUSIONS: Increasing laser power and irradiation, time increased liposome uptake by cells and penetration of peptide drugs across the skin in a dose-dependent manner. High-energy CO2 fractional laser overcomes the rate-limiting barrier function of the stratum corneum. Further investigations are required to establish the safety and efficacy of fractional laser-irradiation assisted delivery of liposome-encapsulated drugs as a transcutaneous drug delivery system. Lasers Surg. Med. 49:525-532, 2017. © 2016 Wiley Periodicals, Inc.

Estimation of the shielding ability of a tungsten functional paper for diagnostic x-rays and gamma rays.

Tungsten functional paper (TFP) is a novel paper-based radiation-shielding material. We measured the shielding ability of TFP against x-rays and gamma rays. The TFP was supplied in 0.3-mm-thick sheets that contained 80% tungsten powder and 20% cellulose (C6 H10 O5 ) by mass. In dose measurements for x-rays (60, 80, 100, and 120 kVp), we measured doses after through 1, 2, 3, 5, 10, and 12 TFP sheets, as well as 0.3 and 0.5 mm of lead. In lead equivalence measurements, we measured doses after through 2 and 10 TFP sheets for x-rays (100 and 150 kVp), and 0, 7, 10, 20, and 30 TFP sheets for gamma rays from cesium-137 source (662 keV). And then, the lead equivalent thicknesses of TFP were determined by comparison with doses after through standard lead plates (purity >99.9%). Additionally, we evaluated uniformity of the transmitted dose by TFP with a computed radiography image plate for 50 kVp x-rays. A single TFP sheet was found to have a shielding ability of 65%, 53%, 48%, and 46% for x-rays (60, 80, 100, and 120 kVp), respectively. The lead equivalent thicknesses of two TFP sheets were 0.10 ± 0.02, 0.09 ± 0.02 mmPb, and of ten TFP sheets were 0.48 ± 0.02 and 0.51 ± 0.02 mmPb for 100 and 150 kVp x-rays, respectively. The lead equivalent thicknesses of 7, 10, 20, and 30 sheets of TFP for gamma rays from cesium-137 source were estimated as 0.28, 0.43, 0.91, and 1.50 mmPb with an error of ± 0.01 mm. One TFP sheet had nonuniformity, however, seven TFP sheets provided complete shielding for 50 kVp x-rays. TFP has adequate radiation shielding ability for x-rays and gamma rays within the energy range used in diagnostic imaging field.

A novel radiation protection device based on tungsten functional paper for application in interventional radiology.

Tungsten functional paper (TFP), which contains 80% tungsten by weight, has radiation-shielding properties. We investigated the use of TFP for the protection of operators during interventional or therapeutic angiography. The air kerma rate of scattered radiation from a simulated patient was measured, with and without TFP, using a water-equivalent phantom and fixed C-arm fluoroscopy. Measurements were taken at the level of the operator's eye, chest, waist, and knee, with a variable number of TFP sheets used for shielding. A Monte Carlo simulation was also utilized to analyze the dose rate delivered with and without the TFP shielding. In cine mode, when the number of TFP sheets was varied through 1, 2, 3, 5, and 10, the respective reduction in the air kerma rate relative to no TFP shielding was as follows: at eye level, 24.9%, 29.9%, 41.6%, 50.4%, and 56.2%; at chest level, 25.3%, 33.1%, 34.9%, 46.1%, and 44.3%; at waist level, 45.1%, 57.0%, 64.4%, 70.7%, and 75.2%; and at knee level, 2.1%, 2.2%, 2.1%, 2.1%, and 2.1%. In fluoroscopy mode, the respective reduction in the air kerma rate relative to no TFP shielding was as follows: at eye level, 24.8%, 30.3%, 34.8%, 51.1%, and 58.5%; at chest level, 25.8%, 33.4%, 35.5%, 45.2%, and 44.4%; at waist level, 44.6%, 56.8%, 64.7%, 71.7%, and 77.2%; and at knee level, 2.2%, 0.0%, 2.2%, 2.8%, and 2.5%. The TFP paper exhibited good radiation-shielding properties against the scattered radiation encountered in clinical settings, and was shown to have potential application in decreasing the radiation exposure to the operator during interventional radiology.

Optimization of Couch Modeling in the Change of Dose Calculation Methods and Their Versions.

In external radiotherapy, the X-ray beam passes through the treatment couch, leading to the dose reduction by the attenuation of the couch. As a method to compensate for the reduction, radiation treatment planning systems (RTPS) support virtual couch function, namely "couch modeling method". In the couch modeling method, the computed tomography (CT) numbers assigned to each structure should be optimized by comparing calculations to measurements for accurate dose calculation. Thus, re-optimization of CT numbers will be required when the dose calculation algorithm or their version changes. The purpose of this study is to evaluate the calculation accuracy of the couch modeling method in different calculation algorithms and their versions. The optimal CT numbers were determined by minimizing the difference between measured transmission factors and calculated ones. When CT numbers optimized by Anisotropic Analytical Algorithm (AAA) Ver. 8.6 were used, the maximum and the mean difference of transmission factor were 5.8% and 1.5%, respectively, for Acuros XB (AXB) Ver. 11.0. However, when CT numbers optimized by AXB Ver. 11.0 were used, they were 2.6% and 0.6%, respectively. The CT numbers for couch structures should be optimized when changing dose calculation algorithms and their versions. From the comparison of the measured transmission to calculation, it was found that the CT numbers had high accuracy.

HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

Dosimetric Verification around High-density Materials for External Beam Radiotherapy.

It is generally known that the dose distribution around the high-density materials is not accurate with commercially available radiation treatment planning systems (RTPS). Recently, Acuros XB (AXB) has been clinically available for dose calculation algorithm. The AXB is based on the linear Boltzmann transport equation - the governing equation - that describes the distribution of radiation particles resulting from their interactions with matter. The purpose of this study was to evaluate the dose calculation accuracy around high-density materials for AXB under three X-rays energy on the basis of measured values with EBT3 and compare AXB with various dose calculation algorithms (AAA, XVMC) in RTPS and Monte Carlo. First, two different metals, including titanium and stainless steel, were inserted at the center of a water-equivalent phantom, and the depth dose was measured with EBT3. Next, after a phantom which reproduced the geometry of measurement was virtually created in RTPS, dose distributions were calculated with three commercially available algorithms (AXB, AAA, and XVMC) and MC. The calculated doses were then compared with the measured ones. As a result, compared to other algorithms, it was found that the dose calculation accuracy of AXB at the exit side of high-density materials was comparable to that of MC and measured value with EBT3. However, note that AXB underestimated the dose up to approximately 30% at the plane of incidence because it cannot exactly estimate the impact of the backscatter.

Kinetics of reactions at an interface: functionalisation of silicate glass with porphyrins via covalent bonds.

Porphyrins carrying either a primary alcohol, a tertiary alcohol or a primary bromide linker group were allowed to react with the surface silanol groups on silicate glass thermally at 80-240 °C to obtain a monolayer film. The kinetics of the reaction was analysed based on the pseudo-second order equation. The tertiary alcohol and the primary bromide reacted much slower than the primary alcohol. Arrhenius plots indicated that higher activation energies can account for the slower reaction of both tertiary alcohol and primary bromide linkers. The introduction of six dodecyl chains into hydroxyporphyrin accelerated the anchoring reaction by a factor of 50 owing to the larger frequency factor of the reaction, demonstrating that the dynamics of the interface is one of the dominant factors regulating the reaction kinetics.

Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

[Use of surgical clips to verify positional accuracy in image-guided accelerated partial breast irradiation].

The purpose of this study was to evaluate the accuracy of positional verification during overall radiation treatment periods in accelerated partial breast irradiation using one or more surgical clips. We first investigated the appropriate computed tomography (CT) slice thickness and detectability of clips for a matching criterion in a phantom study. Next, clinical investigations were carried on 12 patients with multiple clips positioned around the lumpectomy cavity. During radiation treatment planning, a 5-mm region of interest (5-mm ROI) was defined by adding a three dimentional (3D) margin of 5 mm to each clip. During treatment, the clips on two orthogonal kilovoltage X-ray images acquired were moved so as to be included in the corresponding 5-mm ROI on digitally reconstructed radiographs (DRRs). Positional accuracy was calculated using the displacement of each clip in the verification images. The displacements of each clip acquired in all setups were then calculated throughout the overall radiation treatment period and the factors affecting the displacement of clips were investigated. Positional accuracy was also investigated in setups using skin marks and in setups using the bone structure around the thorax. We demonstrated in a phantom study that a CT slice thickness of 2.5 mm was appropriate. In our clinical investigations, 91% of the clips were included in the 5-mm ROI. The interfractional displacement of clips was large, with a long distance between the isocenter and each clip at the time of radiation treatment planning.