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Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia. Here we describe a simple method to induce hypoxia in zebrafish embryos and larvae. This protocol is easy and reproducible and does not require expensive equipment or specialized Screening zebrafish larvae images are comprised of many features which could be extracted development and evaluating the effects of the chemical aautomatically or manually. These two types of feature extraction compounds is started from the early ages of the samples. This are applied and analysed for the system purposes

Zebrafish Larvae This zebrafish model of ototoxicity can be employed as a high throughput screening tool to screen compounds with potential ototoxic effect and as a drug discovery tool to screen anti-ototoxic compounds. Keywords: zebrafish, teratogen, ototoxicity, drug discover The activity of zebrafish larvae is often described in terms of velocity, distance moved, mobility, and even angular velocity. All parameters easily measured with video tracking. Commonly, larvae are placed in well-plates, depending on the study, housing up to 96 individuals in one plate for simultaneous video tracking Zebrafish larvae also exhibit a related escape behavior that has a longer onset latency (~30 ms), is less stereotyped than the C-start, and is mediated by the activity of non-Mauthner cell circuits rather than by the Mauthner cells (Burgess and Granato, 2007b; Issa et al., 2011) (but see Liu and Fetcho, 1999). FIGURE 2 Figure 2 The use of early-stage zebrafish for biomedical research spans early organogenesis to free-swimming larva. A key benefit of this model organism is that repeated assessments spanning several days can be performed of individual larvae within a single experiment, often in conjunction with administered drugs The translucent zebrafish larva is an ideal model organism for studying neuronal circuits regulating brain states, owning to the possibility of easy imaging and manipulating activity of genetically identified neurons while the animal performs stereotyped and well-characterized behaviors

Zebrafish are omnivorous, primarily eating zooplankton, phytoplankton, insects and insect larvae, although they can eat a variety of other foods, such as worms and small crustaceans, if their preferred food sources are not readily available. In research, adult zebrafish are often fed with brine shrimp, or paramecia In vivo and in vitro investigations were performed in zebrafish (Danio rerio) larvae and a zebrafish liver cell line (ZFL), respectively, to understand the potential risks and molecular mechanisms of OC toxicity. The 96-h median lethal concentration (LC 50) of OC was determined to be 251.8 μM in larvae and 5.5 μM in ZFL cells Zebrafish larvae show a clear and distinct pattern of swimming in response to light and dark conditions, following the development of a swim bladder at 4 days post fertilization. This swimming behavior is increasingly employed in the screening of neuroactive drugs

Research Brief 302: PAH and Hypoxia Exposure Result in

To establish a mechanism in which zebrafish larvae could be infected, the team initially attempted to inoculate wild-type zebrafish with SARS-CoV-2 by adding viral load to the water of 2 and 4 dpf. Zebrafish larvae and adults were maintained according to standard protocols. All zebrafish, Danio rerio (Hamilton 1922), were bred from wild-type (AB line) colonies housed in a flow-through tank system (Aquatic Habitats, Apopka, FL, USA) that was maintained at 28.5°C on a 14 h:10 h light-dark cycle

2. For zebrafish larvae up to 8-15 dpf euthanasia requires a secondary method in order to ensure death. This age group can survive anesthetic overdose and rapid chilling even after prolonged absence of heartbeat. They can revive if returned to water that is within their normal environmental parameters Zebrafish possess a prominent binocular structure which is the most observable organ among all, especially at the larvae stages, allowing for continuous and non-invasive observation throughout ocular development During the day, zebrafish larvae also dramatically increase their motor activity in response to both light increments and decrements ( 18, 19 ). To determine whether these visual-motor responses (VMRs) are abolished at night, 5-day-old zebrafish larvae were tested with this assay at 11:00 and 2:00 AM

Select a stage name below to get a detailed description and images. You can also browse the zebrafish Anatomical Ontology (AO) to show anatomical terms that are present at that stage. (h = hours of development at 28.5°C Zebrafish Pronephros Development The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals Xu et al. show that the temporal motor sequence for defensive response in zebrafish larvae consists of an initial escape and a subsequent swimming. This sequential movement is initiated by M-cell activation, which drives neurons in bilateral nucleus of medial longitudinal fascicle via activation of interconnected hindbrain cranial relay neurons Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to. Zebrafish larvae is one of the few model organisms where, using fluorescence-based reporters of the cell activity, it is possible to optically reconstruct the neuronal dynamics across the whole..

The zebrafish larva represents an excellent model to investigate the assembly and function of the motor circuits driving jaw movements. By 5 days post-fertilization, the embryonic yolk sac has been depleted and zebrafish larvae begin exogenous feeding for nutrition old zebrafish larvae by HPLC‐MS., Figure S2: The total ion chromatogram (A) of the water:acetoni‐ trile extract from zebrafish larvae with extracted ion chromatograms of serotonin and dopamine in 7‐day‐old zebrafish larvae by HPLC‐MS 10 zebrafish larvae for each treatment were immersed in fish DW containing 10 mg/mL epinephrine (E-4250, Sigma-Aldrich) for about 30 min to count the number of melanophores. Dorsal view images were taken by means of EC3 camera (Leica Microsystem), using the Leica S8 Apo stereomicroscope The locomotion of zebrafish larvae during six alternating cycles of light-dark exposure was slower during light periods and faster during dark periods; 100-300 mg/L caffeine increased and decreased locomotion during light and dark cycles, respectively, with high inter-cycle reproducibility

Hypoxic Treatment of Zebrafish Embryos and Larva

Although image size is an important feature for triggering hunting in zebrafish larvae (Bianco et al., 2011; Romano et al., 2015), distance perception through image size may not be sufficient, because vision in zebrafish is monocular with a fixed focus and so retinal image size is an ambiguous cue for the true size of an object Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies The first diet was a commercial pellet for zebrafish larva (micron, Sera) and was used as a negative control (control); the second diet had fish meal as the principal protein source (100FM) and the third diet contained an inclusion of 50% soybean meal (50SBM) (Table 1)

Zebrafish larvae at 3 days post fertilization (dpf) were used. The transgenic line sensory:GFP was used 24 to visualize all sensory neurons in the developing larvae in a nacre background, lacking.. Introduction. Zebrafish is a well-known fish model used in a growing number of scientific disciplines 1,2 due to its rapid organogenesis and transparent body. 3 The ability to rear zebrafish from egg to adult in the laboratory is of paramount importance, 4 although raising zebrafish from larvae to juveniles can be laborious, requiring frequent water exchanges and a continuous culture of. Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their. Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases

(PDF) Zebrafish Larvae Classification based on Decision

  1. Zebrafish larvae hatch with a notochord and develop a vertebral column several days after starting to swim. (5) These changes in the locomotory system might affect the bending behaviour of the body and the shape of the body wave. (6)The finfold is gradually replaced as paired and unpaired fins develop, which changes the distribution of.
  2. The retina is the thin layer of tissue in the eye that can receive light stimuli and convert them into electric signals to be transmitted to the brain. The cells that sense fine detail cluster at the center of the retina while the motion-sensing cells that keep track of movement lie at the periphery. When zebrafish larvae hunt, their motion-sensing cells are triggered as a prey crosses their.
  3. THE ZEBRAFISH BOOK. A guide for the laboratory use of zebrafish. Danio (Brachydanio) rerio. by Monte Westerfield, Institute of Neuroscience, University of Oregon. GENERAL METHODS FOR ZEBRAFISH CARE. BREEDING. EMBRYONIC AND LARVAL CULTURE. MICROSCOPIC OBSERVATIONS. CELLULAR METHODS
  4. Having confirmed that larval fish are capable of experiencing pain and benefit from pain relief, Sneddon and Lopez-Luna recommend, Larval zebrafish can be used as a model for the study of pain.
Researchers discover potential epilepsy drug using

Zebrafish larvae activity - Zebrafish research Noldu

Frontiers Learning and memory in zebrafish larvae

Zebrafish larvae are an established model for testing vertebrate toxicity, teratogenicity and environmental risk assessment [120, 121, 122]. The use of these models has proven to be a valuable resource for testing the toxicity of various extracts and condensates obtained from tobacco cigarette smoke [ 6 , 123 , 124 ] Xu et al. show that the temporal motor sequence for defensive response in zebrafish larvae consists of an initial escape and a subsequent swimming. This sequential movement is initiated by M-cell activation, which drives neurons in bilateral nucleus of medial longitudinal fascicle via activation of interconnected hindbrain cranial relay neurons A: Zebrafish larvae (6 dpf, n=24/group) were exposed to haloperidol (final concentration in the water: 0 µM/control, black; 2.5 µM, blue; 5 µM, green; 10 µM, red) and motor activity was measured 4 hours later. The upper trace shows illuminance (sinusoidal waveform 20 - 560 lx, period 10 minutes; y-axis) and the lower traces show mean. Zebrafish Larva Eye Distinguishes Between Prey and Predator. Small and large objects activate various circuits in the visual system of zebrafish larvae. This separation begins in the eye and probably decides the direction of the swimming behavior. Credit: Max Planck Institute for Medical Research. A new study from the Max Planck Institute for.

The pen/lgl2 gene is expressed in the entire epidermis of 24-hour-old zebrafish larvae and the expression declines thereafter(Fig. 5A; data not shown). Using cell size as a criterion, we confirmed that lgl2 transcripts are indeed present in the basal cells of the epidermis, including fin fold epidermis Zebrafish Larvae Husbandry. Maintenance and handling of zebrafish followed international consensus protocols (Westerfield, 2000), and the planning and execution of all experiments were compliant with European regulation (European Union, 2010).Adult wild-type AB/TL zebrafish were used to fertilize eggs and kept in glass aquaria (max 6/l, volume 10 l, 120 × 220 × 490 mm; Fleuren & Nooijen BV. Zebrafish embryos are beautifully transparent, which allows us to follow their development from a fertilised cell to a swimming larvae. In the movie 4 embry.. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause.

Delay of Initial Feeding of Zebrafish Larvae Until 8 Days

Neuromodulation and Behavioral Flexibility in Larval

In the zebrafish model, the small larval size and conserved morphology of the pronephros offers a general applicability for high-throughput assays as larvae can easily be treated and screened in the 96-well format as it has been shown before for modulators of polycystic kidney disease using a morpholino-based genetic zebrafish model 24 or a. The Zebrafish International Resource Center is a non-profit organization. The prices we charge for products and services are to offset some of the actual costs we incur. Please acknowledge the Zebrafish International Resource Center in all publications resulting from the materials and/or services we are providing to you. Thank you During the last decade the zebrafish, Danio rerio, has been introduced as an important model for the study of developmental biology.Key advantages of this vertebrate system are that the embryos and early larvae stages are transparent and develop rapidly, that the zebrafish reproduce easily, and that orthologous genes in the zebrafish and humans regulate similar developmental processes. Zebrafish larvae exposed to BuP 500 μg/L and EtP 5000 μg/L revealed increased anxiety-like behaviour in novel environment. Larvae treated with 500 μg/L of BuP showed reduced activity in familiar and marginally in unfamiliar environment, and larvae exposed to 5000 μg/L of EtP exhibited hyperactivity in familiar environment Emotional states such as fear and anxiety are possible to be observed in zebrafish larvae. With a comprehensive behavioral range, these animals are already responsive to the environment at 24 hpf.

Zebrafish - Wikipedi

  1. Zebrafish larvae were placed in 60 mm dishes (50 larvae per dish) and were exposed to hypoxia or cold stress at 96 hpf. To investigate the development of cross-resistance to hypoxia and cold, zebrafish larvae were first acclimated to 5% O2 at 28°C (pre-hypoxia) or 18°C in air (pre-cold) for 24 h, the controls were maintained in air at 28°C
  2. Justifications. OLAW offers the following justification for maintaining oversight of zebrafish larvae immediately after hatching. The PHS Policy III.A. defines an animal as any live, vertebrate animal used or intended for use in research, research training, experimentation, or biological testing or for related purposes. Although zebrafish embryos develop anatomical structures.
  3. Zebrafish larvae help in search for appetite suppressants. In the study the researchers used zebrafish as a novel testing system for psychoactive substances. Credit: National Institute of Genetics.
  4. Moreover, zebrafish larvae also internalize PS-NPs, accumulating them in the gut and pancreas. However, at concentrations of up to 50 mg l −1 in an acute exposure (48 h), PS-NPs do not interfere with the survival of the larvae after a lethal bacterial challenge ( Aeromonas hydrophila )
  5. zebrafish or its environmental preferences, and there is a possibility that some may be being housed under sub-optimal conditions. Efforts to better define ideal standards relating to the husbandry, care and use of zebrafish are only now beginning (Obenschain & Aldrich 2007)
  6. e level in zebrafish larvae, which is might be the reason for the cardiac physiology changes observed after saccharine exposure. View Full-Text. Keywords: artificial sweeteners; zebrafish.

Zebrafish larvae tissue sections were illuminated with polarized and brightfield light on a Leica BM6000 microscope. White balanced images were captured with a × 63 [1.2 numerical aperture (N.A.)] water immersion objective and a Leica DFC450C 5-megapixel CCD color camera We investigate the effect of these microorganisms on the microbiome and neutrophil response (inflammation) in zebrafish larvae line Tg(Bacmpx:GFP)i114. We postulated that preinoculation of larvae with yeasts would attenuate the intestinal neutrophil response and prevent modification of the larval microbiome induced by the pathogen Valproic acid (VPA) is widely used as an antiepileptic drug or mood stabilizer. In this study, we evaluated the effects of treatment with 2 mM VPA for 3 h on cell proliferation in the telencephalic area of zebrafish larvae using bromodeoxyuridine (BRDU) to label dividing cells. It was demonstrated that 2 mM VPA exposure for 3 h at 2 and 3 days post-fertilization (dpf) larvae decreased cell. In an era of global antibiotic resistance, Willis et al. characterize the living-antibiotic action of predatory Bdellovibrio bacteria in zebrafish larvae versus the human pathogen Shigella flexneri. Results are proof of principle that predators assist the immune system to promote animal survival upon infection by Gram-negative pathogens Zebrafish embryos and larvae were exposed to DMSO or MEHP from 6 to 120 hpf, and gene expression was assessed at 15 dpf. Data are presented as mean fold change normalized to the mean of housekeeping genes 18 s, b2m, and beta actin ± SEM. Data were analyzed using two-way ANOVA with Tukey's multiple comparisons

Integrated zebrafish research | zebrafishlab

For triple staining of concanavalin A (ConA), Na + pump, and H + pump, live larvae were preincubated in zebrafish solution containing 0.5 mg/ml Texas red-conjugated ConA (Molecular Probes, Eugene, OR) for 10 min. After being washed in normal zebrafish solution for 3 min, the ConA-labeled larvae were fixed and immunostained as described above. 2.2. Oil Red O Staining. Zebrafish larvae of each group were collected and fixed with 4% paraformaldehyde (PFA) overnight at 4°C, washed 3 times with phosphate-buffered saline (PBS), and infiltrated sequentially with 20%, 40%, 80%, and 100% propylene glycol (Sigma, USA) at room temperature for 15 min, respectively

Toxic effects of octocrylene on zebrafish larvae and liver

  1. Streptococcus iniae causes systemic infection characterized by meningitis and sepsis. Here, we report a larval zebrafish model of S. iniae infection. Injection of wild-type S. iniae into the otic vesicle induced a lethal infection by 24 h postinfection. In contrast, an S. iniae mutant deficient in polysaccharide capsule (cpsA mutant) was not lethal, with greater than 90% survival at 24 h.
  2. Using zebrafish larvae, we found that the robustness of ChR2-induced locomotor activity was restored after an interval of rest (Wahlstrom-Helgren et al. 2019). However, it is not known how well, or if, ChR2-induced locomotor activity is maintained during repetitive blue- light stimulation over long time periods; a requirement of many.
  3. Zebrafish - Larval Period. The Duration of this period is from 72 hours to 30 days. The Transition from Protruding-mouth to Day 30-44. The pectoral fin continues to develop and the internal organs become more complex
  4. g larvae, feed them with dried brine shrimp powder. Free-swim
  5. ants like GenX
  6. The developing brain is a prime example of an extremely complex network, which makes developmental neurotoxicity one of the most challenging areas in toxicology. We have developed a systems toxicology method that uses a computable biological network to represent molecular interactions in the developing brain of zebrafish larvae
2012 Annual Report of the Division of Intramural Research

Zebrafish - An alternative auditory caused reflexes in the larvae form or young fishes are also performed. In tap produced habituation, the stimulus is normally deliver in normal intervals like 1m for about 10 or more stimulus demonstration.[4] B. Conditioned Place Preference This is a process sometime mixed with self-. Here, we present an FSGS‐like model in zebrafish larvae, an eligible vertebrate model for kidney research. In a transgenic zebrafish strain, podocytes were depleted, and the glomerular response was investigated by histological and morphometrical analysis combined with immunofluorescence staining and ultrastructural analysis by transmission. In this note we describe some new data on successive induction and a simple theory for its operation. MacPhail et al. (2009) measured the movement of individual zebrafish larvae (6 days post-fertilization [dpf]) separately housed in 96-well microtiter plates. The movement (cm/unit time) of each larva as a function of changes in the level of.

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping. Source: NCBI PubMed ( ID PMID:29543903 Larvae up to 14 dpf can be kept in a laboratory space approved by the IACUC. Larvae up to 14dpf will be housed in conditioned clean water. No additional water quality monitoring is necessary for fish up to this age; If unfamiliar with zebrafish husbandry practices please contact an OAR veterinarian for guidance Reference derived and endogenous CD63+ EVs in larval zebrafish. 24,28 Additionally, we have provided the first cryo-EM assessment of total larval and adult zebrafish cardiac EVs, which reveals size distributions reminiscent of EVs from other species and cell types48,49 and highlights the plethora of different EV morphologies found in vivo 2.2 Exposure of zebrafish larvae to Aroclor 1254, α-naphthoflavone, SKF525A, dimethyl sulfoxide and methanol 34 2.3 RNA extraction from whole zebrafish embryos and larvae 35 2.4 The synthesis of cDNA, the polymerase chain reaction (PCR), and quantitative PCR 35 2.5 In vivo fluorescence assays of drug metabolism 37.

Animals in research: zebrafish

Danio rerio commonly known as the Zebrafish is a tropical fish belonging to the minnow family (Cyprinidae), commonly kept in aquaria and used for scientific research. Zebra Danios are of no. ABSTRACT The zebrafish, Danio rerio , has become a popular vertebrate model for the study of infections, mainly because of its excellent optical accessibility at the embryonic and larval stages, when the innate immune system is already effective. We have thus tested the susceptibility of zebrafish larvae to the human pathogen Listeria monocytogenes , a gram-positive, facultative, intracellular. Re: [Zbrafish] On raising Zebrafish larvae. Lawrence, Christian. 9/8/09 7:50 PM. In all likelihood, the fish are basically starving. At 28C, they start exogenous feeding at 5 days, but also continue to derive nutritional benefit from the yolk sac, but this is generally gone by day 7 Systems Toxicology Approach for Assessing Developmental Neurotoxicity in Larval Zebrafish Author: Roman A. Li Subject: Adverse outcomes that result from chemical toxicity are rarely caused by dysregulation of individual proteins; rather, they are often caused by system-level perturbations in networks of molecular events Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate zebrafish larvae exposure metho

Biomedicines Free Full-Text Zebrafish Larvae as a

This research herein presents the construction of a near-infrared optical reflectance microspectroscopy (NIRORM), examining the rhythm of AV valve of wildtype zebrafish larvae in vivo as a preliminary drug toxicity assay, of which the experimental results indicate that NIRORM is effective in discriminating types of cardiac drugs based upon the. Development of cold resistance in zebrafish larvae. To address whether zebrafish larvae are able to build cold-resistance under a mild low temperature, 96 hpf larvae were incubated at 16°C for 24 h followed by severe cold exposure at 12°C and the controls were maintained at 28°C before severe cold exposure at 12°C (Figure 1A). Larvae at 96 hpf were used to perform the experiment because. Zebrafish hindbrain neurons group into distinct mono- and binocular clusters. To localize and functionally characterize hindbrain neurons active during oculomotor behaviour, we stimulated larvae with patterns of moving gratings to elicit optokinetic responses while measuring GCaMP6f calcium signals in individual neurons (Fig. 1a, b). Zebrafish show a high degree of binocular coordination: most. The zebrafish larvae (n = 40 in each) were placed into two identical plastic chambers (x = 5 cm, y = 4 cm, z = 3 cm, kindly shared from the laboratory of H. A. Burgess) filled with 60 mL of fish medium (Fig. 1a). An infrared illuminator (wavelength 600-800 nm) was used to create the recording illumination, and an additional white light was. Zebrafish larvae also continue to be an important model organism in toxicology for both human and environmental hazard assessment (Bambino and Chu 2017; Horzmann and Freeman 2018). Similar to embryos of other teleost fish species, zebrafish embryos are assumed to develop in a sterile environment inside of the chorion, until they hatch at 2 days.

Could Zebrafish larvae be a novel animal model for SARS-CoV-2

Zebrafish larvae evade predators by sensing water flow

Naringenin inhibits alcoholic injury by improving lipid

The Incoherent Fluctuation of Folate Pools and

2. Zebrafish: a perfect experimental animal model. Danio rerio (Hamilton and Buchanan, 1822), commonly known as zebrafish, is a small tropical freshwater fish which lives in river basins of India, Northern Pakistan, Nepal, Bhutan, and South Asia. It belongs to the family of Cyprinidae, within the order of the Cypriniformes. Zebrafish adults are 4-5 cm long, and so they can be easily managed. ZIRC Protocols. protocols/start.txt · Last modified: 2019/11/01 11:46 by rubin The venlafaxine-exposed larvae were less active and covered shorter distance in a light and dark behavioral test compared to the controls. Overall, zygotic exposure to venlafaxine disrupts early development, including brain function, and compromises larval behavior, suggesting impact of this drug on developmental programming in zebrafish In zebrafish, an immature epidermis is established at 24 hpf, formed by the enveloping layer (EVL) and the epidermal basal layer (EBL). Despite numerous descriptions about E-cadh role in epiboly and gastrulation, there is scarce information about E-cadh distribution in the epidermis beyond this stage and during the embryo to larval transition

Zebrafish as a Developmental System

Zebrafish larvae lose vision at night PNA

In larval zebrafish, exposure to dark after a light period results in a characteristic increase in locomotor activity (light-seeking behavior) [43, 44]. A luminescence level of 18 lx for periods of 10 or 20 min follows behavioral protocols used previously [ 4 , 42 , 45 ] Fin-Tail-Coordination-during-Escape-and-Predatory-Behavior-in-Larval-Zebrafish-pone.0032295.s004.ogv 7.8 s, 268 × 188; 578 KB FMIB 48829 Stages in the development of the larva of Terebratulina septentrionalis.jpeg 824 × 263; 22 K How to feed a zebrafish. It is difficult to raise zebrafish for the fact that zebrafish have special needs when it comes to food. Baby and adult zebrafish have different diets, so one has to be aware of the age of the zebrafish when it comes to feeding. There are three types of zebrafish food: Paramecium, Brine Shrimp, and flake food. Parameciu Zebrafish are vertebrates with high genetic similarity to humans that offer external development, high fecundity, and larval transparency and reach sexual maturity quickly, allowing large-scale studies that were not previously possible in mammalian models

Sorting dead from live zebrafish embryos - YouTubeZebrafish video tracking - EthoVision XT | Noldus