Memorization and reproduction of songs in nightingales (Luscinia megarhynchos): evidence for package formation

Summary To examine the properties of avian song memorization, handraised nightingales (Lusdnia megarhynchos) were exposed to three strings composed of 21 or 12 different song types (Fig. 1) which they heard only during the tutoring programs. The analysis of the birds' singing performed several months later revealed a kind of song memorization and production which, hitherto, had not been studied in song birds and is called package formation.Each package was a temporally consistent group of acquired song types which could be traced back to a coherent succession of, usually, three to five (max. seven) model song types. Model song types which a bird failed to acquire were located outside those sequences of model song types that had been acquired and later formed the packages. In contrast to the order of the tutored strings, packages were sung in a variable order. In parallel, neither the frequency of occurrence of the packages nor the performance length was related to the size of the package.We infer from these results that package formation reflects basic properties of the nightingales' song-type memories, which — through segmentation of serially presented learning stimuli — may facilitate the acquisition of large signal repertoires.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

Song recognition memory in juvenile zebra finches: effects of varying the number of presentations of heterospecific and conspecific songs

Seven male and three female zebra finches were exposed to 14 zebra finch (CON) and 14 starling (HET) songs during their sensitive period for song learning and then tested for their recognition memory of both the CON and HET songs in two separate memory tests. Amount of song exposure was varied by presenting individual songs either 3, 9, 27, or 81 times per day for nine consecutive days. After song exposure the birds were trained to discriminate two of the exposed, familiar songs (FAM) from two novel songs (NOV) in a go/no-go operant discrimination procedure, with FAM songs as "go" stimuli. Following discrimination training, untrained FAM and NOV songs were presented as probe songs without reinforcement. Birds responded more to FAM than NOV songs at all levels of song exposure, indicating that the songs were recognized. There were no differences in recognition memory for CON and HET song at any level of song exposure. The results suggest that selective song learning does not result from selective memorization of conspecific song.     

Neuroanatomical evidence for indirect connections between the medial preoptic nucleus and the song control system: possible neural substrates for sexually motivated song

In European starlings (Sturnus vulgaris) as in other seasonally breeding songbirds, a major function of song during the breeding season is mate attraction, and song in this context is highly sexually motivated. Song learning, perception, and production are regulated by nuclei of the song control system, but there is no evidence that these nuclei participate in the motivation to sing. Evidence suggests that the medial preoptic nucleus (POM), a diencephalic nucleus outside of the song control system, might regulate the motivation to sing, at least in a sexual context. If the POM is involved in the regulation of sexually motivated song, then this structure must interact with the song control system. To examine possible neuroanatomical connections between the POM and song control nuclei a tract-tracing study was performed in male starlings using the antero- and retrograde tract tracer, biotinylated dextran amine (BDA). No direct connections were identified between the POM and song control nuclei; however, labeled fibers were found to terminate in a region bordering dorsal-medial portions of the robust nucleus of the archistriatum (RA). Additionally, several indirect routes via which the POM might communicate with the song control system were identified. Specifically, POM projected to dorsomedial nucleus intercollicularis (DM), mesencephalic central gray (GCt), area ventralis of Tsai (AVT), and locus ceruleus (LoC), structures projecting directly to nuclei involved in song production (DM vocal-patterning and respiratory nuclei; GCt, AVT, LoC RA and HVC, and the context in which song is sung (AVT area X). These results are consistent with the possibility that the POM regulates sexually motivated song through interactions with brain regions involved in vocal production.We gratefully acknowledge grant sponsors NIMH (R01-MH 65645) to LVR and NSF for a graduate research fellowship to SJA     

How songbirds deal with large amounts of serial information: retrieval rules suggest a hierarchical song memory

Many songbirds develop remarkably large vocal repertoires, and this has prompted questions about how birds are able to successfully learn and use the often enormous amounts of information encoded in their various signal patterns. We have studied these questions in nightingales (Luscinia megarhynchos), a species that performs more than 200 different types of songs (strophen), or more than 1000 phonetically different elements composing the songs. In particular, we investigated whether and how both song repertoires and song performance rules of nightingales were coded by auditory stimuli presented in serial learning experiments. Evaluation of singing episodes produced by our trained birds revealed that nightingales cope well with an exposure to even long strings of master song-types. They can readily acquire information encoded within and between the different master songs, and they memorize, for example, which master song-types they have experienced in the same learning context. Imitations of such song-types form distinct sequential associations that are termed “context groups”. Additionally, nightingales develop other song-type associations that are smaller in size and termed “package groups”. Package formation results from constraints of the acquisition mechanisms which obviously lead to a segmentation of auditorily perceived master song sequences. Further experimentation validated that the song memory of nightingales is organized in a hierarchical manner and holding information about “context groups” composed of packages, “package groups” composed of songs, and songs composed of song elements. The evidence suggests that implementation of such a hierarchical organization facilitates a quick retrieval of particular songs, and thereby provides an essential prerequisite for a functionally appropriate use of large vocal repertoire is in songbirds. Received: 4 October 1997 / Accepted in revised form: 26 August 1998     

Three models of song learning: Evidence from behavior

Research on avian song learning has traditionally been based on an instructional model, as exemplified by the sensorimotor model of song development. Several large-scale, species-wide field studies of learned birdsongs have revealed that variation is narrowly restricted to certain aspects of song structure. Other aspects are sufficiently stereotyped and so widely shared by species' members that they qualify as species-specific universals. The limitations on natural song variation are difficult to reconcile with a fully open, instructive model of song learning. An alternative model based on memorization by selection postulates a system of innate neural templates that facilitate the recognition and rapid memorization of conspecific song patterns. Behavioral evidence compatible with this model includes learning preferences, rapid conspecific song learning, and widespread ocurrence of species-specific song universals that are recognized innately but fail to develop in songs of social isolates. A third model combines instruction, in the memorization phase, with selection during song production. An overproduced repertoire of plastic songs previously memorized by instruction is winnowed by selection imposed during social interactions at the time of adult song crystallization. Selection during production is well established as a factor in the song development of several species, in the form of action-based learning. The possible role of selective processes in song memorization merits further neurobiological investigation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 501–516, 1997     

Photosynthetic phosphorylation

A brief history of the discovery of photosynthetic phosphorylation by chloroplasts and bacterial chromatophores is presented. Arnon early introduced the terminology of Cyclic and Non-cyclic photophosphorylation and Cyclic and Non-Cyclic electron transport to the processes observed in illuminated chloroplasts. He made major contributions to the elucidation of these processes and stressed their great biological significance. Investigations of the electron transport components of chromatophores have led to the isolation, purification and crystallization of bacterial reaction centers. The development of three-dimensional molecular structures, and the characterization of their electron transfer components have provided a great deal of information about the early reactions of bacterial photosynthesis. The electron transfer schemes presented clearly support the cyclic nature of light-induced electron transfer. Recent developments in the understanding of ATP synthesis in oxidative phosphorylation by mitochondria and in photophosphorylation by chloroplasts and bacterial chromatophores are discussed.Abbreviations ADP, ATP adenosine 5-di- and triphosphates - NADP⁺, NADPH oxidized and reduced Nicotinamide-adenine dinucleotide phosphate - RC reaction center - EPR electron paramagnetic resonance - F₀F₁ ATP-synthase (synthetase) of mitochondria, chloroplasts, and of chromatophores - F₀ membrane portion of ATP-synthase - F₁-ATPase water soluble sector of ATP-synthase     

Neural response to bird’s own song and tutor song in the zebra finch field L and caudal mesopallium

Neurons in the song system nuclei of songbirds exhibit a strong preference for the sound of the birds own song relative to that of conspecific songs. This selectivity is observed in the high vocal center and the nucleus interface of the nidopallium, two song nuclei that receive input from the birds auditory system. To investigate the role of the auditory system in generating the selective responses observed in the song system, we recorded auditory responses in the zebra finch primary auditory forebrain, field L, and in a secondary auditory area, the caudal mesopallium. Field L and caudal mesopallium project directly or indirectly to the high vocal center and nucleus interface of the nidopallium and are presumed to provide substantial auditory input to the song system. We found that, on average, neurons in field L and caudal mesopallium did not show positive selective responses for the birds own song or tutor song relative to conspecific song. Moreover, there were no particular sub-areas in the auditory telencephalon that were relatively more selective than the average. The selectivity for the birds own song would therefore be restricted to song nuclei and would arise in one processing step, potentially found at the interface between the auditory and the song systems.Abbreviations BOS birds own song - CM caudal mesopallium (older term: caudal hyperstriatum ventrale or cHV) - Con conspecific song - HVC high vocal center - LMAN lateral magnocellular nucleus of the anterior nidopallium (older term: lateral magnocellular nucleus of the anterior neostriatum) - LPS pallial-subpallial lamina (older term: lamina medularis dorsalis or LMD) - NCM caudal medial nidopallium (older term: caudo-medial neostriatum) - NIf nucleus interface of the nidopallium (older term: nucleus interface of the neostriatum) - RA robust nucleus of arcopallium (older term: robust nucleus of the archistriatum) - Rev reverse BOS - Revorder reverse order of BOS - Uva nucleus uvaeformis of the thalamusNew avian brain terminology has been used in this paper (). Older terms are given in parentheses in the list of abbreviations     

Oligoadenylates formation on an oligouridylate template in the presence of a lead catalyst

Summary Oligouridylates with more than eight chain units can serve as a template for the template-directed condensation of ImpA catalyzed by Pb²⁺ ion. The templates and the Pb²⁺ ion catalyst facilitate the formation of longer oligoadenylates with five or more units. The ratio of 3–5 linked oligomers to the 2–5 isomers increases with increasing chain length of the oligouridylate template. Short oligouridylates up to a hexamer tend to decrease the yield of oligoadenylates, and do not affect the selectivity of internucleotide linkage.Abbreviations EDTA ethylenediaminetetracetic acid - Tris tris(hydroxymethyl)aminomethane - A adenosine - ImpA adenosine 5-phosphorimidazolide - pA adenosine 5-phosphate - Ap adenosine 2(3)-phosphate - poly A polyadenylic acid - AppA P₁,P₂-diadenosine 5-diphosphate - pAp adenosine 2(3),5-diphosphate - ApA adenylyl adenosine - (pA)_n (n = 2,3,) oligomers of pA - ImpApA 5-phosphorimidazolide of ApA - U uridine - pU uridine 5-phosphate - Up uridine 2(3)-phosphate - poly U polyuridylic acid - pUp uridine 2(3),5-diphosphate - (pU)_n (n = 2,3,) oligomers of pU - (pU)_n – (pA)_m cooligomers composed of (pU)_n and (pA)_m units - AppUpUpUpUp pyrophosphate derived from pA and (pU)₄ - AppUp P₁-(adenosine 5)-P₂-(uridine 2(3)-phosphate 5) -pyrophosphate - BAP bacterial alkaline phosphatase - VPD venom phosphodiesterase - N.P₁ nuclease P₁ - RNase A pancreatic ribonuclease - A^* radioactive adenosine     

The acoustic near field of a dancing honeybee

Summary The acoustic near field close to honeybees performing the wagging dance was investigated with pairs of small, matched microphones placed in various positions around the dancing bees. The dance sounds are produced by the wings, which act as an asymmetrical dipole emitter. Close to the abdomen, the sound pressures in the air spaces above and below the plane of the wings are totally out of phase. A zone of very intense acoustical short-circuiting exists close to the edges of the wings, where pressure gradients of about 1 Pa/mm are observed in the dorso-ventral direction (perpendicular to the plane of the wings). The pressure gradients drive air movements with velocity amplitudes up to about 1 m/s. The pressure gradients are much smaller in directions radially away from the bee and decrease rapidly with increasing distance from the wings. The sound pressure detected by a stationary probe at one side of the bee is strongly modulated at 12–13 Hz as a result of the bee's side-to-side wagging. Surprisingly little sound is found near the dancer's head. The positions of the follower bees reflect the properties of the acoustic field: The follower bees place their antennae in the zone of maximum acoustical short-circuiting where the air particle movements are most intense. These observations suggest 1) how follower bees can avoid mixing up the messages carried by the dance sounds when two or more bees are dancing only a few cm apart and 2) how the followers might extract information about a dancer's spatial orientation from the acoustic near field she produces. The observations also provide clues regarding the nature of the putative sound receivers.Dedicated to Professor Dr. Drs. h.c. mult. H. Autrum on the occasion of his 80th birthday     

The effect of humidity and light on cellular water relations and diffusion conductance of leaves ofTradescantia virginiana L.

Turgor (_p) and osmotic potential (_s) in epidermal and mesophyll cells, in-situ xylem water potential (-xyl) and gas exchange were measured during changes of air humidity and light in leaves ofTradescantia virginiana L., Turgor of single cells was determined using the pressure probe. Sap of individual cells was collected with the probe for measuring the freezing-point depression in a nanoliter osmometer. Turgor pressure was by 0.2 to 0.4 MPa larger in mesophyll cells than in epidermal cells. A water-potential gradient, which was dependent on the rate of transpiration, was found between epidermis and mesophyll and between tip and base of the test leaf. Step changes of humidity or light resulted in changes of epidermal and mesophyll turgor (_p-epi, _p-mes) and could be correlated with the transpiration rate. Osmotic potential was not affected by a step change of humidity or light. For the humidity-step experiments, stomatal conductance (g) increased with increasing epidermal turgor.g/_p-epi appeared to be constant over a wide range of epidermal turgor pressures. In light-step experiments this type of response was not found and stomatal conductance could increase while epidermal turgor decreased.Symbols E transpiration - g leaf conductance - w leaf/air vapour concentration difference - -epi water potential of epidermal cells - -mes water potential of mesophyll cells - -xyl water potential of xylem - _p-epi turgor pressure of epidermal cells - _p-mes turgor pressure of mesophyll cells - _s-epi osmotic potential of epidermal cells - _s-mes osmotic potential of mesophyll cells     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Choosing materials for photographic colorimetry of sectioned tissue

Synopsis The technique of photographic colorimetry depends on the production of blue prints from negatives using a specially formulated developer. This colour is eluted and the absorbance of the eluate gives a measure of the density of colour in the paper. Given that there is a linear relationship between these densities and the densities of the negative used, and that there is linearity between these negative densities and the densities of the stained material photographed, the absorbance of the blue eluates will give a measure of the stain in the material photographed. Different negative densities were simulated by a series of time exposures for a uniform field in an enlarger, using a range of photographic papers. The absorbance values per gram of paper were determined and plotted against the logarithm of the exposure time. A paper is useful when this is a linear relationship. Kodak Kodabromide, contrast grade 2, gave the plot with the longest linear section. Kodak Poly-contrast and Ilford Ilfobrom were similar but Agfa-Gevaert Bromvira and Kodak Medallist had lower absorbance values for equivalent exposures and showed limited linearity. Four Kodak films, High-Contrast Copy (HC), Panatomic X (Pan X), Plus X (PX) and Super XX (XX) were used to photograph polyacrylamide films containing Feulgen-stained DNA. The absorbance of these films was known and thus could be related to the absorbance of their blue images. PX and XX were the best general purpose emulsions. The HC emulsion was sensitive to exposure conditions and was linear in this response only over a short range of Feulgen absorbance values.     

A monoclonal antibody chromosome marker analysis used to locate a loose smut resistance gene in wheat chromosome 6A

Many genes have been located in wheat chromosomes, yet little is known about the location of genes for resistance to Ustilago tritici, which causes loose smut. Crosses were made between the loose smut susceptible alien substitution lines Cadet 6Ag(6A) and Rescue 6Ag(6A) (lines in which Agropyron chromosome 6 is substituted by wheat chromosome 6A) and four cultivars resistant to U. tritici race T19: Cadet, Kota, Thatcher and TD18. The segregating progeny were tested for reaction to race T19 and for the level of binding with a monoclonal antibody specific to a chromosome 6A-coded seed protein. The antibody, which does not bind to seed protein extracts in the absence of the 6A chromosome, was used as a chromosome marker. An association was established between resistance to race T19 and the presence of chromosome 6A for each of the cultivars tested, indicating that resistance to race T19 resides in chromosome 6A. Ustilago tritici race T19 resistance in Cadet appears to be located in the short arm of chromosome 6A, based on the evaluation of the Cadet 6A long ditelosomic stock, which was susceptible, and the Cadet 6A-short: 6-Agropyron- short alien translocation stock, which was resistant.     

Amino acid sequence of the alpha- and beta-globin chains of the Hiroo sea snake (Laticauda laticaudata)

We determined the hemoglobin complete amino acid sequences of the Hiroo sea snake (Laticaudia laticuada) from the intact globin chain, enzymatically digested fragments, and chemical cleavage fragments to analyze molecular evolution for classification of the sea snake. The Hiroo sea snake has two hemoglobin components, Hb-I and Hb-II, which contain different - and -chains, respectively. This is the first report of the complete primary structure of a snake hemoglobin. The sequences were compared with those of other reptilian hemoglobins. Amino acid replacements at positions critical for structure and physiological role of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors at -globins seemed to be fullfilled.     

A conceptual model of ecosystem restoration triage based on experiences from three remote oceanic islands

A conceptual model, that illustrates restoration, ecological landscaping, rehabilitation and regreening, is developed. It considers biocentric, historical, aesthetic and engineering aspects. The term ecosystem restoration triage is used because the first step is to decide whether to do nothing (because, on the one hand, the system is too degraded to warrant restoration, or, on the other, because biological integrity is relatively intact and therefore either none, or minimal, restoration is required) or to do something (because restoration is worthwhile, urgent and feasible). This approach hinges on the definition that restoration in the strictist sense is a biocentric activity that returns the original compositional, structural and functional diversity, along with its dynamics and natural evolutionary potential. Original is a difficult qualifier as it depends on just how far back in time we go. Where human values are involved, this is not restoration in the pure sense of restoring ecological integrity, but is ecological landscaping, rehabilitation or regreening. Experience from three remote oceanic islands [Easter Island, Cousine Island (Seychelles), Marion Island (Sub-Antarctic)] and which represent near extremes of this model are used to illustrate it.     

Post-translational proteolytic processing and the isolectins of lentil and other Viciae seed lectins

Electrospray mass spectrometry was used to identify precisely the proteolytic cleavage points within, and at the C-termini of, the proprotein forms of four Viciae lectins that give rise to their two-chain forms. The lectins examined were the pea and lentil lectins, favin and theLathyrus odoratus lectin, which represent each of the four genera in this tribe. The molecular mass data showed single -chain forms for each lectin, with masses consistent with the available sequence and glycopeptide data, indicating that each came from a single proprotein. In contrast, the pea, lentil andL. odoratus -chains occurred in as many as five forms, due to multiple C-terminal cleavage points. Only favin showed a single -chain form. The -chain mass data were again consistent with the sequence information available, except for the lenti lectin -chain which was re-determined by protein sequencing. The two isolectin forms of this protein were shown to arise from -chain species with and without residue Lys53. The mass spectrum of concanavalin A was also examined and both the single-chain form and the two fragment forms showed no evidence of C-terminal heterogeneity.     

Sexual preferences for songs in female domestic canaries (<Emphasis Type="Italic">Serinus canaria</Emphasis>): can late song exposure,without social reinforcement,influence the effects of early tutoring?

We studied the relative effects of early and late song exposure without social reinforcement on female sexual preferences in the domestic canary (Serinus canaria). Young female canaries were tape-tutored during their first 4 months of life with songs of either domestic or wild male canaries (DT and WT conditions). When they reached sexual maturity, these females were placed in breeding conditions and some of them were re-exposed to songs. During this late exposure the females, according to their experimental group, were either presented with new domestic or wild songs (DL and WL conditions) over 40 days, or were kept without song stimulation (– condition). Afterwards, we assessed the sexual preferences of all the females for domestic or wild songs using the copulation solicitation display assay. The results showed that both DT/– and DT/DL females showed a clear preference for domestic songs. However, whereas WT/WL females preferred wild songs; WT/– females did not show any preference. Finally, DT/WL and WT/DL females failed to show any preference. It appeared that a second song experience at the beginning of their first breeding season, without any social reinforcement, allowed the emergence or stabilisation of early preferences, or interfered with these early preferences depending on whether the song category used during the late exposure phase matched or not the song category used during the early tutoring phase, and also depending on which category was used during the first tutoring phase. This behavioural plasticity could help young adult females to adjust the standard they built during infancy to new environmental conditions.Communicated by P.K. McGregor     

The involvement of oxygen-derived free radicals in the resistant response of potato tubers toErwinia carotovora

Summary The absolute requirement of oxygen for a potato tuber (cv. Pentland Crown) to display a resistant response toErwinia carotovora has been demonstrated in EPR measurements. These show that a relatively stable free radical is formed in inoculated hosts only after exposure to air. In an attempt to isolate and identify unstable free radical precursors, experiments have been conducted in which the chemical spin trap -(4-pyridyl 1-oxide)-N-tert-butyl-nitrone (POBN) was incorporated into the tissue either at the time of inoculation or upon exposure to air. A single radical adduct was observed with spectral parameters that resemble those of a lipid-derived radical.     

Field performance of lines derived from haploid and diploid tissues of Hordeum vulgare

Summary Plant tissue culture technology is of increasing interest to plant breeders. As part of a continuing investigation into breeding methods with spring barley two studies were conducted to assess the field performance of the progenies of material regenerated in tissue culture. The first study involved two spring barley cultivars, Golden Promise and Mazurka and compared lines produced from immature embryo (IE) derived callus with those from embryos developed by the Hordeum bulbosum (Hb) technique of chromosome elimination. In general the mean values for the seven characters scored were lower for the IE than the Hb material. In the second study F₁ hybrid material (Golden PromisexMazurka) was used and doubled haploid lines produced by the H. bulbosum and microspore culture (M) techniques were compared with single seed descent (SSD) material. Analysis of these F samples indicated that the mean values for the M lines were significantly lower than those of the Hb and SSD lines. Furthermore, data from the M lines showed significant evidence of variation created during the culture phase. The implications of these findings for barley breeding are discussed.