Unusual semi-extractability as a hallmark of nuclear body-associated architectural noncoding RNAs.

NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi-extractability. A comparison of RNA-seq data from needle-sheared and control samples revealed the existence of multiple semi-extractable RNAs, many of which were localized in subnuclear granule-like structures. The semi-extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion-like domain of the RNA-binding protein FUS This observation suggests that tenacious RNA-protein and protein-protein interactions, which drive nuclear body formation, are responsible for semi-extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.

Detection of viral components in exosomes derived from NDV-infected DF-1 cells and their promoting ability in virus replication.

Exosomes are micro messengers encapsulating RNA, DNA, and proteins for intercellular communication associated with various physiological and pathological reactions. Several viral infection processes have been reported to pertain to exosomal pathways. However, because of the difficulty in obtaining avian-sourced exosomes, avian virus-related exosomes are scarcely investigated. In this study, we developed a protein A/G-correlated method and successfully obtained the Newcastle disease virus-related exosome (NDV Ex). These exosomes promoted NDV propagation, proven by both GW4869-mediated deprivation and exosomal supplementation. Viral structural proteins NP and F were detected in the NDV Ex and further investigation indicated that the NP protein can be transferred to DF-1 cells through exosomes. The intracellular NP protein exhibited viral replication-promoting and cytokine-suppressing abilities. Therefore, NDV infection produces exosomes, which transfer viral NP protein and promote NDV infection, emphasizing the importance of exosomes in an NDV infection.

Protective Effects of Hydrolyzed Nucleoproteins from Salmon Milt against Ethanol-Induced Liver Injury in Rats.

Dietary nucleotides play a role in maintaining the immune responses of both animals and humans. Oral administration of nucleic acids from salmon milt have physiological functions in the cellular metabolism, proliferation, differentiation, and apoptosis of human small intestinal epithelial cells. In this study, we examined the effects of DNA-rich nucleic acids prepared from salmon milt (DNSM) on the development of liver fibrosis in an in vivo ethanol-carbon tetrachloride cirrhosis model. Plasma aspartate transaminase and alanine transaminase were significantly less active in the DNSM-treated group than in the ethanol plus carbon tetrachloride (CCl4)-treated group. Collagen accumulation in the liver and hepatic necrosis were observed histologically in ethanol plus CCl4-treated rats; however, DNSM-treatment fully protected rats against ethanol plus CCl4-induced liver fibrosis and necrosis. Furthermore, we examined whether DNSM had a preventive effect against alcohol-induced liver injury by regulating the cytochrome p450 2E1 (CYP2E1)-mediated oxidative stress pathway in an in vivo model. In this model, CYP2E1 activity in ethanol plus CCl4-treated rats increased significantly, but DNSM-treatment suppressed the enzyme's activity and reduced intracellular thiobarbituric acid reactive substances (TBARS) levels. Furthermore, the hepatocytes treated with 100 mM ethanol induced an increase in cell death and were not restored to the control levels when treated with DNSM, suggesting that digestive products of DNSM are effective for the prevention of alcohol-induced liver injury. Deoxyadenosine suppressed the ethanol-induced increase in cell death and increased the activity of alcohol dehydrogenase. These results suggest that DNSM treatment represents a novel tool for the prevention of alcohol-induced liver injury.

Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein.

The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged N- and C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.

Comparison of different ELISA protocols for the detection of IgA against influenza nucleoproteins in trachea of vaccinated chickens.

Vaccines targeting mucosal immunity are important for the control of infection by pathogens with mucosal portals of entry, such as avian influenza. However, reliable and effective methods for determining levels of mucosal IgA stimulated by vaccination are not well developed in poultry and are necessary for determining efficacy. The objective of the present study was to compare different ELISA protocols to evaluate levels of mucosal IgA against two different sequences of nucleoprotein (NP:), a highly conserved internal protein in avian influenza virus, in trachea. Positive control tracheas were obtained through hyperimmunization of birds with adjuvated NP1 and NP2 peptide conjugated with keyhole limpet hemocyanin administered both orally and parenterally; negative birds received no antigen. Trachea samples were homogenized, and supernatant fluid was collected to separate IgA. ELISA was performed on NP1- or NP2-positive trachea samples, negative trachea samples, and blank wells with different levels of NP1 and NP2 coating peptides (5 or 10 µg/mL) using two different secondary antibodies (Gene Tex, GT:, or Thermo Scientific, TS:), with or without an acetate wash, and using maximum, medium, or low binding ELISA plates. The TS antibody resulted in a higher background signal compared to GT. Furthermore, coating plate wells with NP2 resulted in very high background compared to NP1. An acetate buffer wash resulted in the muffling of signals, and medium and low binding plates used in the study resulted in better results than maximum binding plates. These results suggest that the selection of appropriate secondary antibodies, binding plates, and ELISA reagent protocols all play important roles in determining NP1- or NP2-specific IgA levels in trachea samples.

Identification of RNA partners of viral proteins in infected cells.

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of protein-protein and RNA-protein interactions within a cell to achieve efficient replication and spreading. Deciphering these interactions is essential to reach a comprehensive understanding of the viral infection process. To study RNA-protein complexes directly in infected cells, we developed a new approach based on recombinant viruses expressing tagged viral proteins that were purified together with their specific RNA partners. High-throughput sequencing was then used to identify these RNA molecules. As a proof of principle, this method was applied to measles virus nucleoprotein (MV-N). It revealed that in addition to full-length genomes, MV-N specifically interacted with a unique population of 5' copy-back defective interfering RNA genomes that we characterized. Such RNA molecules were able to induce strong activation of interferon-stimulated response element promoter preferentially via the cytoplasmic pattern recognition receptor RIG-I protein, demonstrating their biological functionality. Thus, this method provides a new platform to explore biologically active RNA-protein networks that viruses establish within infected cells.

Neurological lesions in chickens experimentally infected with virulent Newcastle disease virus isolates.

Distribution, character, and severity of lesions were evaluated in tissues from the central nervous system of chickens inoculated with 10 different Newcastle disease virus (NDV) isolates: CA 1083, Korea 97-147, Australia (all velogenic viscerotropic), Texas GB and Turkey North Dakota (both velogenic neurotropic), Nevada cormorant, Anhinga and Roakin (all mesogenic), and B1 and QV4 (lentogenic). Tissues for the present study included archived formalin-fixed, paraffin-embedded brain (all strains) plus spinal cord (two strains). Encephalitis was observed in all velogenic viscerotropic and velogenic neurotropic strains, and in some mesogenic strains. In general, the encephalitic lesions began at 5 days post infection, with more severe lesions occurring around 10 days post infection. At this time point, especially in the grey matter of the brain, cerebellum and spinal cord, there were neuronal necrosis, neuronal phagocytosis, and clusters of cells with microglial morphology. Axonal degeneration and demyelination was also observed. Immunohistochemistry (IHC) for viral nucleoprotein confirmed the presence of virus. In the areas of encephalomyelitis, IHC for CD3 revealed that many of the inflammatory cells were T lymphocytes. IHC using an antibody for glial fibrillar acid protein showed reactive astrogliosis, which was most pronounced at the later time points.

Magnetic Particle Spectroscopy for Detection of Influenza A Virus Subtype H1N1.

Magnetic nanoparticles (MNPs) with proper surface functionalization have been extensively applied as labels for magnetic immunoassays, carriers for controlled drug/gene delivery, tracers and contrasts for magnetic imaging, etc. Here, we introduce a new biosensing scheme based on magnetic particle spectroscopy (MPS) and the self-assembly of MNPs to quantitatively detect H1N1 nucleoprotein molecules. MPS monitors the harmonics of oscillating MNPs as a metric for the freedom of rotational process, thus indicating the bound states of MNPs. These harmonics can be readily collected from nanogram quantities of iron oxide nanoparticles within 10 s. The H1N1 nucleoprotein molecule hosts multiple different epitopes that forms binding sites for many IgG polyclonal antibodies. Anchoring IgG polyclonal antibodies onto MNPs triggers the cross-linking between MNPs and H1N1 nucleoprotein molecules, thereby forming MNP self-assemblies. Using MPS and the self-assembly of MNPs, we were able to detect as low as 44 nM (4.4 pmole) H1N1 nucleoprotein. In addition, the morphologies and the hydrodynamic sizes of the MNP self-assemblies are characterized to verify the MPS results. Different MNP self-assembly models such as classical cluster, open ring tetramer, and chain model as well as multimers (from dimer to pentamer) are proposed in this paper. Herein, we claim the feasibility of using MPS and the self-assembly of MNPs as a new biosensing scheme for detecting ultralow concentrations of target biomolecules, which can be employed as rapid, sensitive, and wash-free magnetic immunoassays.

Triple gene block protein interactions involved in movement of Barley stripe mosaic virus.

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells.

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.

Isolation and electrophoretic analysis of nucleoli, phenol-soluble nuclear proteins, and outer cyst walls from Acanthamoeba castellanii during encystation initiation.

A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. Thes nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.

Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy.

The interphase nucleus of the normal rat hepatocyte has been studied in ultrathin frozen sections after glutaraldehyde fixation and the modification of various staining procedures known to be specific for DNA structures (Moyne's thallium stain, Gautier's osmium-ammine) or preferential for RNP carriers and basic proteins (regressive stains based on the use of EDTA or citrate, negatively charged colloidal iron). The results are comparable to those obtained after classical dehydration and embedding. Particular attention has been paid to the nucleolus and extranucleolar RNP components, such as perichromatin fibrils and granules, as well as interchromatin granules. A striking observation was the uneven size and the strongly increased number of perichromatin granules, and the appearance of a contiguous interchromatin net, containing nucleoproteins. Cryoultramicrotomy without embedding appears to be very useful for the exploration of the nucleus in thick sections which remain sufficiently transparent even with the usual accelerating voltages.

Fractionation of the nuclear matrix. I. Partial separation into matrix protein fibrils and a residual ribonucleoprotein fraction.

Isolated rat liver nuclear matrices have been partially separated by means of mild sonication into a matrix protein (matricin) fraction and a residual ribonucleoprotein (RNP) fraction. The initial matricin fraction is composed largely of protein (91.1%) but also contains significant amounts of DNA (8.4%). Reconstruction experiments indicate that this DNA is not the result of the artifactual binding of DNA to the matrix during the extraction procedures. Subsequent treatment with DNase I results in purified matricin composed of greater than 99.5% protein. SDS acrylamide gel electrophoresis of the matrix protein fibrils reveals only three bands: the primary matrix polypeptides of 62,000, 66,000, and 70,000 daltons. Electron microscopy demonstrates a diffuse reticulum with fibrils as thin as 30--50 A and the presence of 80--100-A globular structures. The residual RNP fraction is composed largely of protein (80.1%) and RNA (19.5%), with only traces of DNA (1.1%). Over 98% of the total matrix-associated RNA is recovered in this fraction. SDS acrylamide gel electrophoresis indicates an enrichment in both low and high molecular weight secondary matrix polypeptides, although the 60,000--70,000-dalton polypeptides are present in significant amounts as well. Ultrastructural analysis of the residual RNP fraction reveals distinct electron-dense-staining matrix particles (150--350 A) attached to a fibrous matricin network.

Movement of a karyophilic protein through the nuclear pores of oocytes.

It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to identify the specific sites within the envelope through which transport occurs and determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from approximately 20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 A (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.

Isolation of giant silk fibroin polysomes and fibroin mRNP particles using a novel ribonuclease inhibitor, hydroxystilbamidine.

Hydroxystilbamidine isethionate, a dye capable of binding to both DNA and RNA, has been found to be a powerful inhibitor of cellular ribonucleases. A procedure has been developed that, with the aid of this compound, permits the preparative isolation of giant silk fibroin polyribosomes from the posterior silk gland of Bombyx mori. The polyribosomes contain approximately 45-112 ribosomal particles, as judged by electron microscopy. Treatment of giant fibroin polyribosomes with EDTA releases a particle that sediments at 125S. This mRNP particle contains biologically active silk fibroin mRNA, as judged by cell-free translation in an mRNA-dependent reticulocyte cell-free system.

Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae.

We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases. CEN3 DNA is organized in chromatin such that a 220-250-bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encoding the Bam HI-recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984). Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete structural and functional centromeric unit. A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI. The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.

A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones.

We have detected and begun to characterize a 17-kD centromere-specific protein, CENP-A (Earnshaw, W. C., and N. Rothfield, 1985, Chromosoma., 91:313-321). Sera from several humans with CREST scleroderma autoimmune disease (CREST: calcinosis, Raynaud's phenomenon, esophageal dsymotility, sclerodactyly, and telangiectasia) bind this protein in immunoblot assays of HeLa whole cell or nuclear extracts. We have affinity purified the anti-17-kD centromere protein (anti-CENP-A) specific antibodies from immunoblots of HeLa nuclear protein. The antibodies react with epitopes present on CENP-A derived from humans but apparently do not recognize specific epitopes in either rat or chicken nuclei. Only human nuclear protein is CENP-A positive by immunoblot. Furthermore, human cells show localization of anti-CENP-A antibody to centromeres by immunofluorescence microscopy, whereas rat cells do not. On extraction from the nucleus, CENP-A copurifies with core histones and with nucleosome core particles. We conclude that this centromere-specific protein is a histone-like component of chromatin. The data suggest that CENP-A functions as a centromere-specific core histone.

Early changes in the synthesis of nuclear and cytoplasmic proteins are induced by nerve growth factor in differentiating rat PC12 cells.

Differentiation of rat pheochromocytoma PC12 cells into neuron-like cells was induced by nerve growth factor (NGF) and changes in the apparent rate of synthesis of cellular proteins were analyzed. Attention was particularly focused on the first few hours of exposure to NGF before significant neurite outgrowth was detectable. Cultures were pulse-labeled for 1-h periods with [35S]methionine and proteins were extracted from various subcellular fractions and analyzed by one-dimensional gradient and two-dimensional equilibrium and nonequilibrium gel electrophoresis. The results showed that although the general level of protein synthesis remained constant, by 8 h NGF increased the apparent rate of synthesis of approximately 11 cytoplasmic and 5 nuclear proteins. For several of these proteins, the effect was apparently NGF-specific, since no induction was observed in dibutyryl cAMP-treated cells. Of interest was the following observation: of the five nuclear proteins, NGF increased the synthesis of two proteins with MrS of 56,000 [doublet] and 50,000 D that were associated with a biochemically and morphologically defined nuclear matrix fraction. A cytoplasmic protein, with an Mr of 92,000 D (pI 4.8) appeared to be induced de novo by NGF. NGF also decreased the rate of synthesis of several cytoplasmic and nuclear proteins of low molecular mass (less than 40,000 D). Since only 1-h pulses of [35S]methionine were used, and since experiments with actinomycin D showed that most of these NGF-induced early changes in rates of synthesis included a transcription-dependent step, it seems likely that early effects of NGF include activation of specific genes.

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Assembly of protein and nucleoprotein particles from extracted tobacco rattle virus protein and RNA.

Protein extracted from tobacco rattle virus by treatment with formic or acetic acid reassociated in vitro to form rings and tubular structures of indeterminiate lengths in 0.25 molar glycine at pH 7.5 to 9.5. When tobacco rattle virus RNA that had been extracted with phenol was incubated with the protein at 9 degrees C, particles with more regular lengths formed; these particles correspond in size and sedimentation properties to the short particles that normally accompany infection by tobacco rattle virus. The biological activity of the reconstitutted short particles was identical to that of native short particles.