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Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ., (© 2021 Shi et al.)

Importance: 131 I-metaiodobenzylguanidine ( 131 I-mIBG) has a significant targeted antitumor effect for neuroblastoma. However, currently there is a paucity of data for the use of 131 I-mIBG as a "front-line" therapeutic agent in those patients with newly diagnosed high-risk neuroblastoma as part of the conditioning regimen for myeloablative chemotherapy (MAC)., Objective: To evaluate the feasibility of upfront consolidation treatment with 131 I-mIBG plus MAC and hematopoietic stem cell transplantation (HSCT) in high-risk neuroblastoma patients., Methods: A retrospective, single-center study was conducted from 2003-2019 on newly diagnosed high-risk neuroblastoma patients without progressive disease (PD) after the completion of induction therapy. They received 131 I-mIBG infusion and MAC followed by HSCT., Results: A total of 24 high-risk neuroblastoma patients were enrolled with a median age of 3.0 years at diagnosis. After receiving this sequential consolidation treatment, 3 of 13 patients who were in partial response (PR) before 131 I-mIBG treatment achieved either complete response (CR) ( n = 1) or very good partial response (VGPR) ( n = 2) after HSCT. With a median follow-up duration of 13.0 months after 131 I-mIBG therapy, the 5-year event-free survival and overall survival rates estimated were 29% and 38% for the entire cohort, and 53% and 67% for the patients who were in CR/VGPR at the time of 131 I-mIBG treatment., Interpretation: Upfront consolidation treatment with 131 I-mIBG plus MAC and HSCT is feasible and tolerable in high-risk neuroblastoma patients, however the survival benefit of this 131 I-mIBG regimen is only observed in the patients who were in CR/VGPR at the time of 131 I-mIBG treatment., Competing Interests: No financial or nonfinancial benefits have been received or will be received from any party related directly or indirectly to the subject of this article., (© 2020 The Authors. Pediatric Investigation published by John Wiley & Sons Australia, Ltd on behalf of Futang Research Center of Pediatric Development.)

Enhanced telomere maintenance is evident in malignant cancers. While telomeres are thought to be inherently heterochromatic, detailed mechanisms of how epigenetic modifications impact telomere protection and structures are largely unknown in human cancers. Here we develop a molecular tethering approach to experimentally enrich heterochromatin protein HP1α specifically at telomeres. This results in increased deposition of H3K9me3 at cancer cell telomeres. Telomere extension by telomerase is attenuated, and damage-induced foci at telomeres are reduced, indicating augmentation of telomere stability. Super-resolution STORM imaging shows an unexpected increase in irregularity of telomeric structure. Telomere-tethered chromo shadow domain (CSD) mutant I165A of HP1α abrogates both the inhibition of telomere extension and the irregularity of telomeric structure, suggesting the involvement of at least one HP1α-ligand in mediating these effects. This work presents an approach to specifically manipulate the epigenetic status locally at telomeres to uncover insights into molecular mechanisms underlying telomere structural dynamics.

A subset of tumors use a recombination-based alternative lengthening of telomere (ALT) pathway to resolve telomeric dysfunction in the absence of TERT. Loss-of-function mutations in the chromatin remodeling factor ATRX are associated with ALT but are insufficient to drive the process. Because many ALT tumors express the mutant isocitrate dehydrogenase IDH1 R132H, including all lower grade astrocytomas and secondary glioblastoma, we examined a hypothesized role for IDH1 R132H in driving the ALT phenotype during gliomagenesis. In p53/pRb-deficient human astrocytes, combined deletion of ATRX and expression of mutant IDH1 were sufficient to create tumorigenic cells with ALT characteristics. The telomere capping complex component RAP1 and the nonhomologous DNA end joining repair factor XRCC1 were each downregulated consistently in these tumorigenic cells, where their coordinate reexpression was sufficient to suppress the ALT phenotype. RAP1 or XRCC1 downregulation cooperated with ATRX loss in driving the ALT phenotype. RAP1 silencing caused telomere dysfunction in ATRX-deficient cells, whereas XRCC1 silencing suppressed lethal fusion of dysfunctional telomeres by allowing IDH1-mutant ATRX-deficient cells to use homologous recombination and ALT to resolve telomeric dysfunction and escape cell death. Overall, our studies show how expression of mutant IDH1 initiates telomeric dysfunction and alters DNA repair pathway preferences at telomeres, cooperating with ATRX loss to defeat a key barrier to gliomagenesis. Significance: Studies show how expression of mutant IDH1 initiates telomeric dysfunction and alters DNA repair pathway preferences at telomeres, cooperating with ATRX loss to defeat a key barrier to gliomagenesis and suggesting new therapeutic options to treat low-grade gliomas. Cancer Res; 78(11); 2966-77. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Effective communication in health information plays an important role in health promotion and cancer prevention. Cancer-related information acquisition can happen via active and purposeful seeking, but may also happen less purposely via the routine use of media and interactions with other people (called scanning). We examined seeking and scanning behaviors regarding cancer prevention in older Chinese adults, identified commonly used sources of information of such behaviors, and examined their associations with fatalistic beliefs and cancer fear. A convenience sample of 224 community-dwelling adults aged ≥60 were recruited between May and July in 2013 in Hong Kong. Results suggested that cancer information scanning (79.5%) was more common than information seeking (30.4%) among our participants. Health professional was the most popular source for both scanning (78.7%) and seeking (58.8%) behaviors regarding cancer information. Fatalistic beliefs was significantly and negatively associated with seeking behaviors (OR = 0.50) but not scanning behaviors, and cancer fear showed no relationship with either behavior. This study shows that the cancer information seeking and scanning behaviors were still suboptimal in this age group and adds to the knowledge regarding the associations between fatalistic beliefs and fear with cancer information seeking and scanning behaviors among older Chinese adults., Competing Interests: The authors declare no conflict of interest.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1 mut ) genes as they progressed through their lifespan. IDH1 mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1 WT or IDH1 mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1 mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1 mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1 mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR., Competing Interests: There are no relationships that could be construed as resulting in an actual, potential, or perceived conflict of interest with regard to this manuscript., (©2016 American Association for Cancer Research.)

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR., (©2015 American Association for Cancer Research.)

The shortening of telomeres as a causative factor in ageing is a widely discussed hypothesis in ageing research. The study of telomere length and its regenerating enzyme telomerase in the longest-lived non-colonial animal on earth, Arctica islandica, should inform whether the maintenance of telomere length plays a role in reaching the extreme maximum lifespan (MLSP) of >500years in this species. Since longitudinal measurements on living animals cannot be achieved, a cross-sectional analysis of a short-lived (MLSP 40years from the Baltic Sea) and a long-lived population (MLSP 226years Northeast of Iceland) and in different tissues of young and old animals from the Irish Sea was performed. A high heterogeneity of telomere length was observed in investigated A. islandica over a wide age range (10-36years for the Baltic Sea, 11-194years for Irish Sea, 6-226years for Iceland). Constant telomerase activity and telomere lengths were detected at any age and in different tissues; neither correlated with age or population habitat. Stable telomere maintenance might contribute to the long lifespan of A. islandica. Telomere dynamics are no explanation for the distinct MLSPs of the examined populations and thus the cause of it remains to be investigated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bivalve species with exceptional longevity are newly introduced model systems in biogerontology to test evolutionarily conserved mechanisms of aging. Here, we tested predictions based on the oxidative stress hypothesis of aging using one of the tropical long-lived sessile giant clam species, the smooth giant clam (Tridacna derasa; predicted maximum life span: >100 years) and the short-lived Atlantic bay scallop (Argopecten irradians irradians; maximum life span: 2 years). The warm water-dwelling giant clams warrant attention because they challenge the commonly held view that the exceptional longevity of bivalves is a consequence of the cold water they reside in. No significant interspecific differences in production of H2O2 and O2- in the gills, heart, or adductor muscle were observed. Protein carbonyl content in gill and muscle tissues were similar in T derasa and A i irradians. In tissues of T derasa, neither basal antioxidant capacities nor superoxide dismutase and catalase activities were consistently greater than in A i irradians. We observed a positive association between longevity and resistance to mortality induced by exposure to tert-butyl hydroperoxide (TBHP). This finding is consistent with the prediction based on the oxidative stress hypothesis of aging. The findings that in tissues of T derasa, proteasome activities are significantly increased as compared with those in tissues of A i irradians warrant further studies to test the role of enhanced protein recycling activities in longevity of bivalves.

The organization of molecules into macromolecular (nanometer scale), supramolecular complexes (submicron-to-micron scale), and within subcellular domains, is an important architectural principle of cellular biology and biochemistry. Determining the precise nature and distribution of complexes within the cellular milieu is a challenging biophysical problem. Time-series analysis of laser scanning confocal microscopy images by image correlation spectroscopy (ICS) or fluctuation moments methods provides information on aggregation, flow, and dynamics of fluorescently tagged macromolecules. All the methods to date require a brightness standard to relate the experimental data to absolute aggregation. In this article, we show that ICS as a function of gradual photobleaching is a sensitive indicator of aggregation distribution on the submicron scale. Specifically, in photobleaching ICS, the extent of nonlinearity of the apparent cluster density as a function of bleaching is related to the size of clusters. The analysis is tested using computer simulations on model aggregate systems and then applied to an experimental determination of Aβ peptide aggregation on nerve cells. The analysis reveals time-dependent increases in Aβ1-42 peptide aggregation. Globally, the datasets could be described by a monomer-dimer-tetramer-hexamer or a monomer-dimer-trimer-pentamer model. The results demonstrate the utility of photobleaching with ICS for determining aggregation states on the supramolecular scale in intact cells without the requirement for a brightness standard., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Telomere overhangs are essential for telomere end protection and telomerase extension, but how telomere overhangs are generated is unknown. Leading daughter strands synthesized by conventional semiconservation DNA replication are initially blunt, while lagging daughter strands are shorter by at least the size of the final RNA primer, which is thought to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. We show that the final lagging RNA primer is not terminal but is randomly positioned ~70-100 nucleotides from the ends and is not removed for more than an hour. This identifies an important intrinsic step in replicative aging. Telomeric termini are processed in two distinct phases. During the early phase, which occupies 1-2 h following replication of the duplex telomeric DNA, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2, when the C-terminal nucleotide is specified-referred to as the late phase. These observations suggest the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes.

Unlabelled: Thermal sensation is studied experimentally under mixing ventilation, displacement ventilation, and stratum ventilation in an environmental chamber. Forty-eight subjects participated in all tests under the same boundary conditions but different ventilation methods in the classroom. Thermal comfort analysis was carried out according to the designated supply airflow rate, room temperature, and relative humidity for the three ventilation methods. The thermal neutral temperature under stratum ventilation is approximately 2.5 °C higher than that under mixing ventilation and 2.0 °C higher than that under displacement ventilation. This result indicates that stratum ventilation could provide satisfactory thermal comfort level to rooms of temperature up to 27 °C. The energy saving attributable to less ventilation load alone is around 12% compared with mixing ventilation and 9% compared with displacement ventilation., Practical Implications: The confirmation of the significantly elevated thermal neutral temperature can have a number of implications for both thermal comfort in an air-conditioned room and energy consumption of the associate air-conditioning system. With respect to the former, it provides scientific basis for the feasibility of elevated room temperatures, and with respect to the latter, it reveals considerable potentials for energy saving., (© 2011 John Wiley & Sons A/S.)

Telomeres are thought to be maintained by the preferential recruitment of telomerase to the shortest telomeres. The extension of the G-rich telomeric strand by telomerase is also believed to be coordinated with the complementary synthesis of the C strand by the conventional replication machinery. However, we show that under telomere length-maintenance conditions in cancer cells, human telomerase extends most chromosome ends during each S phase and is not preferentially recruited to the shortest telomeres. Telomerase rapidly extends the G-rich strand following telomere replication but fill-in of the C strand is delayed into late S phase. This late C-strand fill-in is not executed by conventional Okazaki fragment synthesis but by a mechanism using a series of small incremental steps. These findings highlight differences between telomerase actions during steady state versus nonequilibrium conditions and reveal steps in the human telomere maintenance pathway that may provide additional targets for the development of anti-telomerase therapeutics.

We have developed a technique to replica plate mammalian cells grown on plastic dishes using low melt agarose. This method is simpler than previously described methods that use polyester membranes to grow and transfer cells. We have tested the effectiveness of this technique on normal and immortal cell lines and have found that we can transfer cells with an efficiency of 80-90%. We have used this technique to rapidly screen clones for insertion of a lentivirally encoded gene without a selectable marker.

The severe acute respiratory syndrome (SARS) crisis led to the construction of a negative pressure operating theatre at a hospital in Hong Kong. It is currently used for treatment of suspected or confirmed airborne infection cases, and was built in anticipation of a return of SARS, an outbreak of avian influenza or other respiratory epidemics. This article describes the physical conversion of a standard positive pressure operating theatre into a negative pressure environment, problems encountered, airflow design, and evaluation of performance. Since entering regular service, routine measurements and observations have indicated that the airflow performance has been satisfactory. This has also been confirmed by regular air sampling checks. Computational fluid dynamics, a computer modelling technique, was used to compare the distribution of room air before and after the design changes from positive to negative pressure. The simulation results show that the physical environment and the dispersion pattern of bacteria in the negative pressure theatre were as good as, if not better than, those in the original positive pressure design.

Background: A negative-pressure operating theater is required to limit the spread of respiratory diseases in patients with severe acute respiratory syndrome, tuberculosis, avian influenza, or similar infectious diseases. In Hong Kong, we converted a conventional operating theater into a negative-pressure operating theater that has been in service for more than a year. In this article, we introduce its ventilation design and evaluate the airflow performance in relation to different combinations of medical lamp configurations and modes of launching infectious particles into the room air., Methods: We used a computational fluid dynamics technique for the numerical analysis., Results: Our analyses showed that the airflow performance in the negative-pressure operating theater was satisfactory and comparable to the original positive-pressure design. The airflow pattern effectively controlled the dispersion of infectious particles. Our calculations demonstrated that the airflow contained the dispersion of infectious particles released from the patient sufficiently to protect the surgical team, and vice versa., Conclusions: Computational fluid dynamics can be used to assess airflow in a negative-pressure operating room and model the dispersion of infectious particles from the patient.

A laminar airflow study was performed in a standard operating theatre in Hong Kong, the design of which followed the requirements of the UK Health Technical Memorandum. The study of the ultra-clean ventilation system investigated the effectiveness of the laminar flow in: (i) preventing bioaerosols released by the surgical staff from causing postoperative infection of the patient; and (ii) protecting the surgical team against infection by bacteria from the wound site. Seven cases of computer simulation are presented and the sensitivity of individual cases is discussed. Air velocity at the supply diffuser has been identified as one of the most important factors in governing the dispersion of airborne infectious particles. Higher velocity within the laminar regime is advantageous in minimizing the heat-dissipation effect, and to ensure an adequate washing effect against particulate settlement. Inappropriate positioning of the medical lamps can be detrimental. Omission of a partial wall may increase the infection risk of the surgical team due to the ingression of room air at the supply diffuser periphery. This paper stresses that a successful outcome in preventing airborne infection depends as much on resolving human factors as on overcoming technical obstacles.

Three groups of double-muscled Belgian Blue young bulls were fed during different stages of production diets differing in the proportions of linolenic and linoleic acid by including linseed in the concentrate or giving grass silage as main linolenic acid suppliers. Samples of rumen and abomasal contents and of the longissimus thoracis, subcutaneous fat, and liver were taken to analyze the fatty acid pattern with emphasis on the individual trans (t) C18:1 fatty acids and cis-9,trans-11 conjugated linoleic acid (c9t11CLA). Trans C18:1 isomers represented up to 20 g/100 g of total fatty acids in rumen and abomasal contents, whereas the accumulation of c9t11CLA was limited. Total trans C18:1 content in subcutaneous fat and intramuscular fat of the longissimus thoracis comprised 8.4 and 5.2 g/100 g of total fatty acids, respectively, with t11C18:1 being the most abundant one. Compared to rumen contents, subcutaneous and intramuscular fat were enriched in c9t11CLA and contained fewer tC18:1 isomers, resulting in a higher c9t11CLA/t11C18:1 ratio (0.04, 0.22, and 0.22, respectively). This result suggests that the endogenous synthesis of c9t11CLA in adipose tissue by the Delta(9)-desaturase was more important than its ruminal production.

Surgical site infection risk due to airborne bacteria is a key area of consideration in developing operating theatre ventilation design and monitoring procedures. This paper reviews the recent extensive research into operating theatre ventilation development in relation to the design concepts in operating theatre layout, pressurization and ventilation, particularly the evolvement of ultra-clean ventilation. The findings that led to the current technical standards and the developments of microbial measurements and numerical techniques are discussed. Since the late 1980s, computational fluid dynamics has been a fast developing tool used in the prediction of room air distribution and contaminant dispersion. The basic principles and current practice applying to operating theatre ventilation studies are introduced.

Grass and grass silage represent a rich and natural source of omega-3 polyunsaturated fatty acids, in particular linolenic acid, for ruminants. Recent research, focusing on improving the content of these beneficial fatty acids in grass, requires storage of the forage samples prior to analysis. In this study, we evaluated whether conservation of fresh grass and grass silage by freezing (1 and 4 weeks,--18 degrees C) and/or drying (24h, 50 degrees C) affected its fatty acid content and induced shifts between lipid classes. FA were extracted using chloroform/methanol (2/1, v/v) and triacylglycerols (TAG), free fatty acids (FFA) and polar lipids (PL) were separated by thin layer chromatography. Fatty methyl esters (FAME) were identified by gas chromatography. Loss of thawing liquor might provoke a dramatic decrease in extractable lipid after frozen storage of both grass and grass silage. Morever, after frozen storage, fatty acids in grass but not in grass silage seem subjected to a higher rate o f lipolysis and oxidation, as suggested by increased quantities of FFA (3.1, 7.6, 8.4 % of total FAME) and reduced proportions of poly-unsaturated fatty acids (79.5, 73.6 and 74.1 % of total FAME) when analysing fresh grass samples directly or after 1 and 4 weeks of frozen storage, respectively. Drying of fresh grass did not provoke changes in FA composition, but distribution of FA over lipid classes was significantly altered, with an increase in TAG (5.1 to 17.9 % of total FAME) and FFA (2.4 to 14.9 % of total FAME) and lower proportions of PL (90.7 to 55.7 % of total FAME).

A group of 80 patients was scanned by ultrasound within 24 hours of insertion of an intrauterine contraceptive device (IUCD) and re-examined clinically and/or by ultrasound after the next menstrual period. An attempt was made to identify factors such as uterine size, uterine flexion and the position of the IUCD which might be related to its expulsion or removal when, on initial scanning, the device was not located in the fundal area; removal was necessary in 5 out of 6 patients. In patients with an endometrial cavity of less than 40 mm length and/or acute uterine flexion, the overall figure for expulsion and removal was 54 per cent (13 out of 24). It is hoped that a consideration of these factors will lead to a reduction of the failure rate of the IUCD.