KORTUC, a novel hydrogen peroxide­based radiosensitizer for the enhancement of brachytherapy in patients with unresectable recurrent uterine cervical cancer.

Kochi Oxydol Radiation Therapy for Unresectable Carcinoma (KORTUC) is a novel radiosensitizer invented by Professor Ogawa at Kochi University (Japan) in 2006. The current study aimed to report the experience of the present authors with the use of KORTUC treatment in combination with interstitial brachytherapy (ISBT), with or without external beam (EB) radiotherapy (RT), in patients with locally recurrent cervical cancer (LRCC), who were likely to have a high risk of poor prognosis. Between April 2012 and January 2020, 14 female patients (15 tumoral lesions) with LRCC underwent KORTUC with ISBT. Their previous treatments included surgery (n=4), radiation therapy (n=8) and surgery plus RT (n=3). The primary lesions were located in the vaginal stump (n=5), pelvic wall (n=3), cervix (n=3), vaginal wall (n=2) and lymph nodes (n=2). At 2 h before RT, KORTUC was injected intratumorally via direct colposcopy. The dose of KORTUC ranged from 4-12 ml, adjusted for the tumor size. For patients who underwent ISBT, KORTUC was administered before and after insertion of the applicator before irradiation. Intratumoral injection of KORTUC was completed without any technical or safety issues in all 15 patients; it was well tolerated with no adverse events observed. KORTUC also showed preferable efficacy; a clinical complete response was observed in 87% of patients and the initial response rate was 100%. The 2-year local control rate in patients who underwent ISBT + KORTUC was 79%, whereas it was 63% in the re-irradiation group which was significantly lower (P=0.02) than that in the non-irradiation group (100%). Based on this finding, KORTUC with external irradiation is considered to be an optimal treatment strategy for patients with newly diagnosed LRCC this disease. Additionally, KORTUC may be an effective radiation response enhancer in multiple cancer types in which locoregional control after RT alone remains poor.

Ca2+ Dependent Formation/Collapse of Cylindrical Ca2+-ATPase Crystals in Scallop Sarcoplasmic Reticulum (SR) Vesicles: A Possible Dynamic Role of SR in Regulation of Muscle Contraction.

[Ca2+]-dependent crystallization of the Ca2+-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.

Helical Three Dimensional Reconstruction Using Bayesian Optimization for Cryogenic Electron Microscopy.

Three-dimensional (3D) reconstruction for cryogenic electron microscopy (cryo-EM) often falls into an ill-posed problem owing to several uncertainties in observations, including noise. To reduce excessive degree of freedom and avoid overfitting, the structural symmetry is often used as a powerful constraint. In the case of the helix, the entire 3D structure is determined by the subunit 3D structure and two helical parameters. There is no analytical method to simultaneously obtain both of the subunit structure and helical parameters. A common approach is to employ an iterative reconstruction in which the two optimizations are performed alternately. However, iterative reconstruction does not necessarily converge when a heuristic objective function is used for each optimization step. Also, the obtained 3D reconstruction highly depends on the initial guess of the 3D structure and the helical parameters. Herein, we propose a method for estimating the 3D structure and helical parameters that also performs an iterative optimization; however, the objective function for each step is derived from a single objective function to make the algorithm convergent and less sensitive to the initial guess. Finally, we evaluated the effectiveness of the proposed method by testing it on cryo-EM images, which were challenging to reconstruct using conventional methods.

Proteomic Analysis Reveals Changes in Tight Junctions in the Small Intestinal Epithelium of Mice Fed a High-Fat Diet.

The impact of a high-fat diet (HFD) on intestinal permeability has been well established. When bacteria and their metabolites from the intestinal tract flow into the portal vein, inflammation in the liver is triggered. However, the exact mechanism behind the development of a leaky gut caused by an HFD is unclear. In this study, we investigated the mechanism underlying the leaky gut related to an HFD. C57BL/6J mice were fed an HFD or control diet for 24 weeks, and their small intestine epithelial cells (IECs) were analyzed using deep quantitative proteomics. A significant increase in fat accumulation in the liver and a trend toward increased intestinal permeability were observed in the HFD group compared to the control group. Proteomics analysis of the upper small intestine epithelial cells identified 3684 proteins, of which 1032 were differentially expressed proteins (DEPs). Functional analysis of DEPs showed significant enrichment of proteins related to endocytosis, protein transport, and tight junctions (TJ). Expression of Cldn7 was inversely correlated with intestinal barrier function and strongly correlated with that of Epcam. This study will make important foundational contributions by providing a comprehensive depiction of protein expression in IECs affected by HFD, including an indication that the Epcam/Cldn7 complex plays a role in leaky gut.

Transposon mutagenesis and genome sequencing identify two novel, tandem genes involved in the colony spreading of Flavobacterium collinsii, isolated from an ayu fish, Plecoglossus altivelis.

Bacteria of the family Flavobacteriaceae (flavobacteria) primarily comprise nonpathogenic bacteria that inhabit soil and water (both marine and freshwater). However, some bacterial species in the family, including Flavobacterium psychrophilum and Flavobacterium columnare, are known to be pathogenic to fish. Flavobacteria, including the abovementioned pathogenic bacteria, belong to the phylum Bacteroidota and possess two phylum-specific features, gliding motility and a protein secretion system, which are energized by a common motor complex. Herein, we focused on Flavobacterium collinsii (GiFuPREF103) isolated from a diseased fish (Plecoglossus altivelis). Genomic analysis of F. collinsii GiFuPREF103 revealed the presence of a type IX secretion system and additional genes associated with gliding motility and spreading. Using transposon mutagenesis, we isolated two mutants with altered colony morphology and colony spreading ability; these mutants had transposon insertions in pep25 and lbp26. The glycosylation material profiles revealed that these mutants lacked the high-molecular-weight glycosylated materials present in the wild-type strain. In addition, the wild-type strains exhibited fast cell population movement at the edge of the spreading colony, whereas reduced cell population behavior was observed in the pep25- and lbp26-mutant strains. In the aqueous environment, the surface layers of these mutant strains were more hydrophobic, and they formed biofilms with enhanced microcolony growth compared to those with the wild-type. In Flavobacterium johnsoniae, the Fjoh_0352 and Fjoh_0353 mutant strains were generated, which were based on the ortholog genes of pep25 and lbp26. In these F. johnsoniae mutants, as in F. collinsii GiFuPREF103, colonies with diminished spreading capacity were formed. Furthermore, cell population migration was observed at the edge of the colony in wild-type F. johnsoniae, whereas individual cells, and not cell populations, migrated in these mutant strains. The findings of the present study indicate that pep25 and lbp26 contribute to the colony spreading of F. collinsii.

Social Motility Assays of Flavobacterium johnsoniae.

Flavobacterium johnsoniae cells move rapidly over solid surfaces by gliding motility. The collective migration of F. johnsoniae on the surfaces results in the formation of spreading colonies. Colony spreading is influenced by adhesin components on the cell surface and the concentrations of agar and glucose. For example, on nutrient-poor agar media, film-like, round spreading colonies are formed. F. johnsoniae displays at least two types of migration: small cell cluster movements leading to concentric colonies and individual cell movements leading to dendritic colonies. The methods for observing colony morphology are described in this chapter.

Elongation and Contraction of Scallop Sarcoplasmic Reticulum (SR): ATP Stabilizes Ca2+-ATPase Crystalline Array Elongation of SR Vesicles.

The Ca2+-ATPase is an integral transmembrane Ca2+ pump of the sarcoplasmic reticulum (SR). Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca2+]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca2+] (≤1.3 µM). The arrays were classified as ''tetramer'', "two-rail" (like a railroad) and ''monomer''. Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca2+] of ~0.002 µM, "two-rail" arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable "two-rail" forms in the presence of ATP at low [Ca2+].

Differentiating Trypanosoma cruzi in a Host Mammalian Cell Imaged in Aqueous Liquid by Atmospheric Scanning Electron Microscopy.

Atmospheric Scanning Electron Microscopy (ASEM) is a powerful tool to observe a wet specimen at high resolution under atmospheric pressure. Here, we visualized a protozoan parasite Trypanosoma cruzi over the course of its infection cycle in the host mammalian cell. This is the first observation of intracellular parasite using a liquid-phase EM. Unlike regular SEM, aldehyde-fixed cell body of T. cruzi appears translucent, allowing the visualization of internal structures such as kinetoplast of trypomastigote and nucleus of amastigote. Plasma membrane of the host mammalian cell also appears translucent, which enabled direct observation of differentiating intracellular parasites and dynamic change of host cellular structures in their near-natural states. Various water-rich structures including micro- and macro- vesicles were visualized around T. cruzi. In addition, Correlative Light and Electron Microscopy exploiting open sample dish of ASEM allowed identification of parasite nucleus and transfected fluorescence-labeled parasites soon after internalization, while location of this morphological intermediate was otherwise obscure. Successful visualization of the differentiation of T. cruzi within the host cell demonstrated here opens up the possibility of using ASEM for observation of variety of intracellular parasites. IMPORTANCE Using Atmospheric Scanning Electron Microscopy (ASEM), we visualized interaction between infectious stage of Trypanosoma cruzi and completely intact host mammalian cell. Plasma membrane appears translucent under ASEM, which not only enables direct observation of T. cruzi within its host cell, but also reveals internal structures of the parasite itself. Sample deformation is minimal, since the specimen remains hydrated under atmospheric pressure at all times. This nature of ASEM, along with the open structure of ASEM sample dish, is suited for correlative light-electron microscopy, which can further be exploited in identification of fluorescent protein in the intracellular parasites.

Biofilm formation of Staphylococcus epidermidis imaged using atmospheric scanning electron microscopy.

Staphylococcus epidermidis are gram-positive bacteria that form a biofilm around implanted devices and develop an infection into a chronic state. Recently, it has been revealed that microvesicles have important roles in biofilm formation and intercellular communication among bacteria. However, biofilm formation of Staphylococcus epidermidis, and its relation to microvesicle secretion, is poorly understood because of the difficulty required to preserve the delicate water-rich morphology of biofilm for high-resolution observations. Here, we successfully imaged the microvesicles secreted from Staphylococcus epidermidis and the subsequent process of their integration into biofilm using liquid-phase imaging using atmospheric scanning electron microscopy (ASEM). In the biofilm, cells were connected by nanotube-like structures attached by microvesicles, and surrounded by extracellular polymeric substances. Cells cultured in the ASEM specimen holder were aldehyde-fixed and stained using positively charged nanogold labelling and/or using National Center for Microscopy and Imaging Research method. The samples immersed in aqueous radical scavenger glucose buffer were imaged by the inverted SEM of ASEM. Information regarding the morphologies of microvesicles, nanotube-like fibrils, and biofilm formed by Staphylococcus epidermidis is expected to be useful to elucidate the biological mechanism of biofilm formation and to develop a medicine against biofilms and their associated infections.

New enzyme-targeting radiosensitizer (KORTUC II) treatment for locally advanced or recurrent breast cancer.

Kochi oxydol radiation therapy for unresectable carcinomas II (KORTUC II) is currently the most widely used radiosensitizer in Japan. This sensitizer is a solution consisting of 0.83% sodium hyaluronate and 0.5% hydrogen peroxide. The mixture is injected intratumorally just before radiation therapy (RT) several times. KORTUC II has the effect of neutralizing antioxidant enzymes, while increasing the oxygen tension into the tumor tissue, and achieves marked local effects without notable adverse events. The present report describes cases in which KORTUC II was used to treat patients with locally advanced breast cancer (LABC) or recurrent breast cancer (LRBC). The present study included 30 patients with LABC (n=9) or LRBC (n=21) aimed at local control of tumors, who were followed up for ≥3 months after treatment. The irradiation dose and extent fields were determined by the attending physicians considering various patient factors, such as a performance status, prognosis and presence or absence of adjuvant therapy. The median irradiation dose was 60.4 Gy3.5 (43.6-76.1 Gy3.5) based on the calculation of equivalents of 2 Gy fractions, and the median total number of sensitizer injections was 5 (2-7) times. The median maximum tumor shrinkage was 97.0% and 15 patients (50%) were assessed to have achieved a clinical complete response. The proportion with loco-regional control at 1, 2 and 3 years was 100, 94.7 and 75.4%, respectively, and progression free survival after RT at 1 and 2 years was 59.0 and 24.1%, respectively. KORTUC II exhibited high rates of local tumor control for LABC and LRBC. KORTUC II is expected to be an inexpensive and promising RT method because it is safe and has an excellent radio-sensitizing effect.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae: 2. Role of Filamentous Extracellular Network and Cell-to-Cell Connections at the Biofilm Surface.

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm.

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm.

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.

Liquid-phase ASEM imaging of cellular and structural details in cartilage and bone formed during endochondral ossification: Keap1-deficient osteomalacia.

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1-/- femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Colony spreading of the gliding bacterium Flavobacterium johnsoniae in the absence of the motility adhesin SprB.

Colony spreading of Flavobacterium johnsoniae is shown to include gliding motility using the cell surface adhesin SprB, and is drastically affected by agar and glucose concentrations. Wild-type (WT) and ΔsprB mutant cells formed nonspreading colonies on soft agar, but spreading dendritic colonies on soft agar containing glucose. In the presence of glucose, an initial cell growth-dependent phase was followed by a secondary SprB-independent, gliding motility-dependent phase. The branching pattern of a ΔsprB colony was less complex than the pattern formed by the WT. Mesoscopic and microstructural information was obtained by atmospheric scanning electron microscopy (ASEM) and transmission EM, respectively. In the growth-dependent phase of WT colonies, dendritic tips spread rapidly by the movement of individual cells. In the following SprB-independent phase, leading tips were extended outwards by the movement of dynamic windmill-like rolling centers, and the lipoproteins were expressed more abundantly. Dark spots in WT cells during the growth-dependent spreading phase were not observed in the SprB-independent phase. Various mutations showed that the lipoproteins and the motility machinery were necessary for SprB-independent spreading. Overall, SprB-independent colony spreading is influenced by the lipoproteins, some of which are involved in the gliding machinery, and medium conditions, which together determine the nutrient-seeking behavior.

Pyrene Excimer-Based Fluorescent Labeling of Cysteines Brought into Close Proximity by Protein Dynamics: ASEM-Induced Thiol-Ene Click Reaction for High Spatial Resolution CLEM.

Fluorescence microscopy (FM) has revealed vital molecular mechanisms of life. Mainly, molecules labeled by fluorescent probes are imaged. However, the diversity of labeling probes and their functions remain limited. We synthesized a pyrene-based fluorescent probe targeting SH groups, which are important for protein folding and oxidative stress sensing in cells. The labeling achieved employs thiol-ene click reactions between the probes and SH groups and is triggered by irradiation by UV light or an electron beam. When two tagged pyrene groups were close enough to be excited as a dimer (excimer), they showed red-shifted fluorescence; theoretically, the proximity of two SH residues within ~30 Å can thus be monitored. Moreover, correlative light/electron microscopy (CLEM) was achieved using our atmospheric scanning electron microscope (ASEM); radicals formed in liquid by the electron beam caused the thiol-ene click reactions, and excimer fluorescence of the labeled proteins in cells and tissues was visualized by FM. Since the fluorescent labeling is induced by a narrow electron beam, high spatial resolution labeling is expected. The method can be widely applied to biological fields, for example, to study protein dynamics with or without cysteine mutagenesis, and to beam-induced micro-fabrication and the precise post-modification of materials.

Network of Palladium-Based Nanorings Synthesized by Liquid-Phase Reduction Using DMSO-H2O: In Situ Monitoring of Structure Formation and Drying Deformation by ASEM.

We developed a liquid-phase synthesis method for Pd-based nanostructure, in which Pd dissolved in dimethyl sulfoxide (DMSO) solutions was precipitated using acid aqueous solution. In the development of the method, in situ monitoring using atmospheric scanning electron microscopy (ASEM) revealed that three-dimensional (3D) Pd-based nanonetworks were deformed to micrometer-size particles possibly by the surface tension of the solutions during the drying process. To avoid surface tension, critical point drying was employed to dry the Pd-based precipitates. By combining ASEM monitoring with critical point drying, the synthesis parameters were optimized, resulting in the formation of lacelike delicate nanonetworks using citric acid aqueous solutions. Precipitation using HCl acid aqueous solutions allowed formation of 500-nm diameter nanorings connected by nanowires. The 3D nanostructure formation was controllable and modifiable into various shapes using different concentrations of the Pd and Cl ions as the parameters.

High-dose-rate interstitial brachytherapy with hypoxic radiosensitizer KORTUC II for unresectable pelvic sidewall recurrence of uterine cervical cancer: a case report.

In order to improve oncologic outcomes in radiotherapy treatments of patients with unresectable pelvic sidewall recurrences of uterine cervical cancer, we combined high-dose-rate interstitial brachytherapy (HDR-ISBT) with newly tested hypoxic radiosensitizer Kochi oxydol-radiation therapy for unresectable carcinomas (KORTUC II), an enzyme-targeting radiosensitization treatment involving intra-tumoral injection of sodium hyaluronate mixed with hydrogen peroxide. We report on a 63-year-old patient referred to our department with an extensive pelvic sidewall recurrence of uterine cervical cancer after initial hysterectomy. The tumor size was 55 × 25 × 80 mm, with a calculated volume of 89.7 cc. Whole pelvic irradiation of 50 Gy in 25 fractions was administered, combined with weekly cisplatin injections. KORTUC II injections were given two times: at day 21 (42 Gy) and at day 24 (48 Gy). After finishing whole pelvic irradiation, HDR-ISBT of 25 Gy in 5 fractions b.i.d. over 3 days was administered. KORTUC II was also injected at the time of implantation. Dose-volume histogram (DVH) values for clinical target volume were D90, D98, and D100 of 6.0, 5.0, and 3.5 Gy per fraction, respectively. D2cc values were 2.1, 4.1, 3.2, and 2.0 Gy per fraction for the bladder, rectum, sigmoid colon, and small bowel, respectively. No acute adverse events ≥ grade 3 were observed. Repeated grade 3 pyelonephritis occurred as a late complication at 11, 24, and 26 months after the treatment, and was successfully resolved with antibiotics. Moreover, grade 2 late toxicity was documented, including sciatic neuralgia, lower limb lymphedema, and urinary incontinence. At present, 32 months after HDR-ISBT, the patient remains free of disease, with no toxicity-related deterioration in physical condition.

Bayesian inference for three-dimensional helical reconstruction using a soft-body model.

Estimation of the three-dimensional (3D) structure of a protein using cryo transmission electron microscopy (cryo-TEM) is an inverse problem, which aims to estimate the parameters of a specific physical process from observations. In general, we need to model the observation process to estimate a structure. However, the inconsistency between the model and a real observation process decreases the estimation accuracy. In cryo-TEM, the flexibility of a soft protein, including the bending of a helix, can lead to inconsistencies between the observations because of the assumption that there is a consistent 3D structure behind each observed image. In this paper, we propose a 3D reconstruction algorithm for helical structures using a parametric soft-body model that can represent continuous deformation. We performed an approximate Bayesian inference for unobservable (hidden) variables, such as the deformation parameters, projection angle, and two-dimensional origin offset (shift) of each protein in the 3D structure estimation problem. Our principled approach is not only beneficial to deal with the uncertainties in the estimation, but also beneficial to make the optimization algorithm convergent and efficient. Reconstructions with artificial molecules validated the advantage of the proposed method, particularly, when deformed helices were imaged under a low signal-to-noise ratio condition. Moreover, we confirmed that the proposed method successfully reconstructed a 3D structure from cryo-TEM images of the tobacco mosaic virus.

Verification of 5-Aminolevurinic Radiodynamic Therapy Using a Murine Melanoma Brain Metastasis Model.

Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, ("radiodynamic therapy (RDT)"), instead of with 5-ALA and laser beams ("photodynamic therapy"), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their ∙OH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.