Fluorescence in situ hybridization data on distances between defined genomic sequences are used to construct a quantitative model for the overall geometric structure of a human chromosome. We suggest that the large-scale geometry during the Go/G1 part of the cell cycle may consist of flexible chromatin loops, averaging -3 million bp, with a random-walk backbone. A fully explicit, threeparametric polymer model of this random-walk/giant-loop structure can account well for the data. More general models consistent with the data are briefly discussed.A human chromosome is a very large molecule. Its DNA strand has in the order of 100 million base pairs (Mbp) arrayed along its contour and has a Mr of -1011 Da. Quantitative information on mammalian chromosome geometry during the interphase part of the cell cycle is very extensive for scales <0.01 Mbp (1, 2) but not for larger scales. At the level of -0.001-0.01 Mbp the DNA is associated with proteins to form a chromatin fiber "30 nm in diameter (1, 2); at scales of -0.1 Mbp the chromatin may form loops (2). Very little is known numerically about the larger-scale geometry, comprising >3 orders of magnitude (0.1-300 Mbp), where the difficulty of following the chromatin fiber as it winds and twists its way within the interphase cell nucleus has crippled quantitative analyses. Yet, large-scale geometric structure of chromosomes influences essential cellular processes such as DNA replication and transcription (2), as well as many specialized functions such as repair or misrepair of ionizing-radiation-produced DNA damage (3, 4).Recently, van den Engh et at (5) used fluorescence in situ hybridization data to quantify some intermediate-scale properties of interphase chromosomes. These investigators measured physical distances between pairs of fluorescently marked specific DNA sequences on human chromosome 4 in fibroblast cells fixed on microscope slides. The observations were made for cells in the Go/G1 phase of the cell cycle, the period between mitosis and the onset of DNA replication. Probe pairs having genomic separations from -0.1 Mbp to "4 Mbp were analyzed. A major conclusion was that, on scales from 0.1 Mbp to 1.5 Mbp, chromatin geometry corresponds to a simple random walk. Observed deviations from random-walk behavior at larger genomic separations could be explained by a polymer model in which the DNA of any one chromosome is confined to a spherical subvolume of the interphase nucleus (6). An alternative suggestion was that the deviations were due to "giant" loops, several Mbp in length (7) (Fig. 1). For values of genomic separation <1.5 Mbp, the points lie approximately on a straight line (Fig. 1A), corresponding to random-walk behavior for chromatin, in agreement with earlier data (5). Genomic sites separated by 10-190 Mbp also show an approximately linear relation but with a much smaller slope (Fig. 1 B). Moreover, for these large genomic separations, the statistical distribution of distances for a given probe pair also corresponds to random-walk behavior (F...

Abstract. We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at N2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales >100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation. SIaORXLY after cell division, the mitotic chromosomes decondense and diffuse into the interphase nucleus. While individual chromosomes cannot be discerned, important processes related to chromosome function take place. Regulatory factors interact with chromatin, DNA is made accessible for transcription, RNA is produced and processed, DNA is replicated, and repairs are made of DNA strand breaks. When the chromosomes reappear for the next mitosis, they have been duplicated and prepared for rapid partitioning over the daughter cells. The complexity of these processes raises many questions about the large-scale organization of chromosomes and how this organization relates to cell function (e.g., Blobel, 1985;Manuelidis and Chen, 1990;Cook, 1991;Lawrence and Singer, 1991;De Boni, 1994).Diverse models, ranging from highly random to highly organized, have been proposed for the higher-order organization of interphase chromatin. These models variously involve irregularly folded fibers (DuPraw, 1965), radial loop structures (Manuelidis and Chen, 1990), giant loops (Ostashevsky and Lange, 1994), semirigid orientation ("Rabl" configuration) (Rabl, 1885;Comings, 1968), or random polymers confined by tethering or external forces (Hahnfeldt et al., 1993). Some models assign to chromo-

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for highthroughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a highresolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible. V C 2015 International Society for Advancement of Cytometry Key terms extracellular vesicle; exosome; microvesicle; microparticle; high-resolution flow cytometry; characterization; sorting; coincidence; swarm; liposome EXTRACELLULAR vesicles (EVs) are small membrane-enclosed vesicles released by cells either by outward budding from the plasma membrane or by the fusion of multivesicular bodies with the plasma membrane resulting in the release of intracellular stored vesicles (1). The release of EVs and their content, i.e., proteins, lipids and RNAs, is tightly regulated and varies not only between different cell types but also depends on the physiological state of the producing cell (2-4). Consequently, EV release is very dynamic and the EV population is very heterogeneous. EVs can function in an autocrine or paracrine fashion, but can also enter the circulatory system and act at distant sites. Hence EVs are present in body fluids like blood, milk, urin...

We have identified three new members of the olfactory receptor (OR) gene family within a large segment of DNA that is duplicated with high similarity near many human telomeres. This segment is present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. Additional copies are present polymorphically at 11 other subtelomeric locations. The frequency with which the block is present at some locations varies among populations. While humans carry seven to 11 copies of the OR-containing block, it is located in chimpanzee and gorilla predominantly at a single site, which is not orthologous to any of the locations in the human genome. The observation that sequences flanking the OR-containing segment are duplicated on larger and different sets of chromosomes than the OR block itself demonstrates that the segment is part of a much larger, complex patchwork of subtelomeric duplications. The population analyses and structural results suggest the types of processes that have shaped these regions during evolution. From its sequence, one of the OR genes in this duplicated block appears to be potentially functional. Our findings raise the possibility that functional diversity in the OR family is generated in part through duplications and inter-chromosomal rearrangements of the DNA near human telomeres.

We demonstrate that members of the olfactory receptor (OR) gene family are distributed on all but a few human chromosomes. Through FISH analysis, we show that OR sequences reside at more than 25 locations in the human genome. Their distribution is biased for terminal bands. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence-relationships are indicative of the inter- and intrachromosomal duplications responsible for OR family expansion. The human genome has accumulated a striking number of dysfunctional copies: 72% of the sequences are pseudogenes. ORF-containing sequences predominate on chromosomes 7, 16 and 17.