Nuclear Localization Peptide Research Articles

Interaction of bifunctional peptide-carbazole complexes with DNA and antimicrobial activity

Two peptide-carbazole conjugates, CTAT and CNLS, were designed and synthesized using carbazole Schiff base to modify the cell membrane penetrating peptide TAT (47–57) and the nuclear localization peptide NLS at the N terminus. The interaction with ctDNA was investigated by multispectral and agarose gel electrophoresis. And the effects of CNLS and CTAT on the G-quadruplex structure were explored by circular dichroism titration experiments. The results show that both CTAT and CNLS interact with ctDNA in a minor groove binding manner. Both conjugates bind more tightly to DNA than the individual substances CIBA, TAT and NLS. In addition, CTAT and CNLS are capable of unfolding parallel G-quadruplex structures and are potential G-quadruplex unfolding agents. Finally, broth microdilution was performed to test the antimicrobial activity of the peptides. The results showed that CTAT and CNLS had a 4-fold increase in antimicrobial activity compared with the parent peptides TAT and NLS. They could exert antimicrobial activity by disrupting the integrity of cell membrane bilayer and binding to DNA, and could be used as novel antimicrobial peptides for the development of novel antimicrobial antibiotics.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP–importin α interactions

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin β and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Å crystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2–ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117–196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors.

Improving the therapeutic index of anticancer agents is an enormous challenge. Targeting decreases the side effects of the therapeutic agents by delivering the drugs to the intended destination. Nanocarriers containing the nuclear localizing peptide sequences (NLS) translocate to the cell nuclei. However, the nuclear localization peptides are nonselective and cannot distinguish the malignant cells from the healthy counterparts. In this study, we designed a "masked" NLS peptide which is activated only in the presence of overexpressed matrix metalloproteinase-7 (MMP-7) enzyme in the pancreatic cancer microenvironment. This peptide is conjugated to the surface of redox responsive polymersomes to deliver doxorubicin and curcumin to the pancreatic cancer cell nucleus. We have tested the formulation in both two- and three-dimensional cultures of pancreatic cancer and normal cells. Our studies revealed that the drug-encapsulated polymeric vesicles are significantly more toxic toward the cancer cells (shrinking the spheroids up to 49%) compared to the normal cells (shrinking the spheroids up to 24%). This study can lead to the development of other organelle targeted drug delivery systems for various human malignancies.

Quantum Dots-Ligand Complex as Ratiometric Fluorescent Nanoprobe for Visual and Specific Detection of G-Quadruplex.

By complexing a nonionic G-quadruplex ligand with hybrid dual-emission quantum dots (QDs), a ratiometric fluorescent nanoprobe is developed for G-quadruplex detection in a sensitive and specific manner. The QDs nanohybrid comprised of a green-emission QD (gQD) and multiple red-emission QDs (rQDs) inside and outside of a silica shell, respectively, is utilized as the signal displaying unit. Only the presence of G-quadruplex can displace the ligand from QDs, breaking up the QDs-ligand complexation, and inducing the restoration of the rQDs fluorescence. Since the fluorescence of embedded gQD stays constant, variations of the dual-emission intensity ratios display continuous color changes from green to bright orange, which can be clearly observed by the naked eye. Furthermore, by utilizing competitive binding of a cationic ligand versus the nonionic ligand toward G-quadruplex, the nanoprobe is demonstrated to be applicable for assessing the affinity of a G-quadruplex-targeted anticancer drug candidate, exhibiting ratiometric fluorescence signals (reverse of that for G-quadruplex detection). By making use of the specificity of the ligand binding with G-quadruplex against a double helix, this nanoprobe is also demonstrated to be capable of sensitive detection of one-base mutation, exhibiting sequence-specific ratiometric fluorescence signals. By functionalizing with a nuclear localization peptide, the nanoprobe can be used for visualization of G-quadruplex in the nucleus of human cells.

Stepwise synthesis of RNA conjugates carrying peptide sequences for RNA interference studies

Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3'-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3'-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.

Targeted silencing of TrkA expression in rat forebrain neurons via the p75 receptor

Basal forebrain neurons express the neurotrophin receptors, p75NTR and tyrosine kinase receptor A (TrkA). We tested the hypothesis that impairment of memory in rats could be achieved by RNA interference (RNAi) –induced silencing of TrkA specifically within these neurons. A novel fusogenic, karyophilic immunoporter (fkAbp75-ipr) was constructed from the antibody, MC192 (monoclonal antibody to the rat neurotrophin receptor p75NTR, Abp75), poly-l-lysine together with the hemagglutinin 2 and VP1 nuclear localization peptides of influenza and SV40 virus, respectively. Plasmid DNA constructs containing short hairpin sequences inhibitory to tyrosine kinase receptor A expression (TrkAi) and the gene encoding cGFP (green fluorescent protein from coral fish) was produced. These TrkAi plasmids were mixed with the immunoporter, forming the immunogene, TrkAi-fkAbp75. A control TrkAsc complexed with fkAbp75 (TrkAsc-fkAbp75) immunogene was constructed from a scrambled sequence (TrkAsc) and fkAbp75-ipr. Rats were infused using an osmotic mini-pump into the third ventricle with either TrkAi-fkAbp75 or TrkAsc-fkAbp75. Naive rats were also included as additional controls. After 7 days, examination of gene expression on forebrain sections of some rats revealed cGFP expression in TrkA neurons. Fifteen to 19 days after infusion, rats were tested in a Morris water maze apparatus. Animals that received TrkAi-fkAbp75 showed significantly impaired spatial memory learning ability compared with naive or TrkAsc-fkAbp75-treated rats. Western blot and immunofluorescence analysis showed that TrkA protein levels and numbers of TrkA positive neurons were reduced by 60% and 55% respectively in TrkAi-fkAbp75-infused rats compared with infused controls or naive animals. We conclude that p75-receptor-mediated RNAi-induced silencing of genes offers a novel and powerful way to study the function of specific endogenous genes within distinct neuronal subpopulations of the brain.

Effects of Mechanical Flexion on the Penetration of Fullerene Amino Acid-Derivatized Peptide Nanoparticles through Skin

Dermatomed porcine skin was fixed to a flexing device and topically dosed with 33.5 mg.mL-1 of an aqueous solution of a fullerene-substituted phenylalanine (Baa) derivative of a nuclear localization peptide sequence (Baa-Lys(FITC)-NLS). Skin was flexed for 60 or 90 min or left unflexed (control). Confocal microscopy depicted dermal penetration of the nanoparticles at 8 h in skin flexed for 60 and 90 min, whereas Baa-Lys(FITC)-NLS did not penetrate into the dermis of unflexed skin until 24 h. TEM analysis revealed fullerene-peptide localization within the intercellular spaces of the stratum granulosum.

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGENE 6 transfection technology. The noncovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a maximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G(2)/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.

Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( approximately 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.

Mouse embryonic stem cells efficiently lipofected with nuclear localization peptide result in a high yield of chimeric mice and retain germline transmission potency

Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 μg DNA for 10 × 10 6 cells via electroporation which resulted in 10–20 positive cells/mm 2, M9-assisted lipofection of 2 × 10 5 cells with 2 μg DNA resulted in > 150 positive cells/mm 2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 × 10 6 ES cells and 6 μg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease.

Novel Nuclear Shuttle Peptide to Increase Transfection Efficiency in Esophageal Mucosal Cells

The major barrier to successful transfection appears to be passage of the DNA plasmid from the cytoplasm into the cell nucleus. The M9 nuclear localization peptide, a fragment of the naturally occurring heterogeneous nuclear ribonucleoprotein A1, which serves to shuttle messenger RNA across the nuclear membrane, has been proposed as a tool for enhancing transfection efficiency. We tested three different reporter plasmids to assess the ability of M9 to improve transfection efficiency in esophageal mucosal cells. The effect of M9 on the intracellular movement of plasmid was also assessed using fluorescent microscopy to trace rhodamine-labeled plasmid. The M9 nuclear shuttle peptide consistently increased the transfection efficiency. When transfection was carried out with specific plasmids, β-galactosidase enzyme activity, keratinocyte growth factor–1 growth factor levels, and the number of transfected cells expressing growth factor peptides were progressively increased with increasing M9 to plasmid ratios. Fluorescent microscopy demonstrated that the M9 shuttle allowed rhodamine-tagged plasmid to gain access to the nucleus, while it was located exclusively in the cytoplasm without the peptide. The M9 shuttle peptide increases transfection efficiency in esophageal mucosal cells, and therefore may have a useful role in gene therapy applications involving the esophagus. ( J Gastrointest Surg 2002;6:37–42.)

Synthesis and binding properties of oligonucleotides carrying nuclear localization sequences.

The synthesis of oligonucleotides carrying nuclear localization peptide sequences is described using two strategies: first, oligonucleotides carrying a thiol group at the 5' end were reacted with maleimido peptides; second, peptide and oligonucleotide were prepared stepwise on the same support, yielding oligonucleotide-3'-peptide conjugates. This second approach was thoroughly studied. Using amino acids and small peptides as model compounds, some side reactions were analyzed, detected, and minimized. Oligonucleotides complementary to Ha-ras gene and carrying nuclear localization peptides at the 3' and 5' ends were prepared. Melting temperature studies showed that duplexes containing nuclear localization peptides were more stable than duplexes with unmodified oligonucleotides. Moreover, oligonucleotide-peptide conjugates maintain a good mismatch discrimination when they bind to their target RNA.

The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells

Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction. Phycoerythrine-NLS transport substrate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methyltransferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenously added cytosolic protein. All the characteristic properties of in vivo nuclear transport are faithfully reproduced with these transport substrates: (i) the transport requires a functional NLS in the transported protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear localization peptide efficiently competes for transport, while the transport-incompetent mutant peptide does not; and (iv) wheat germ agglutinin inhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or inefficient extraction of the cytosolic proteins during the permeabilization of the cells. The nuclear transport was proportional to the number of conjugated NLSs. Nuclear transport of phycoerythrine carrying 7-8 conjugated NLSs/subunit required the addition of exogenous cytosolic proteins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport. Nuclear transport with different combinations of transport substrates further supported the assumption that distinct transport mechanisms operate for different substrates. From a mixture of PE-NLS7-8 and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the absence of exogenous cytosol, while Bsp methyltransferase-NLS was efficiently transported. Exogenous cytosol promoted the nuclear transport of the highly conjugated substrate.

Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses.

Optimal in vitro gene delivery with cationic lipids requires an excess of cationic charges with respect to DNA phosphates. In these conditions, in vivo delivery will be hampered by interference from cationic lipid-binding macromolecules either circulating or in the extracellular matrix. To overcome this problem, we are developing a modular transfection system based on lipid-coated DNA particles reminiscent of enveloped viruses. The particle core consists of the lipopolyamine-condensed nucleic acid in an electrically neutral ratio to which other synthetic lipids with key viral properties are hydrophobically adsorbed. As a first result, we have found that a good transfection level can be achieved simply with the neutral core particle, provided a zwitterionic lipid (dioleoyl phosphatidylethanolamine) is added to completely coat the DNA. Addition of lipids bearing a fusogenic or a nuclear localization peptide head group to the particles does not significantly improve an already efficient system, in contrast to polylysine-based gene transfer methods that rely on lysosomotropic or fusogenic agents to be effective. This emphasizes the distinctive properties of the lipopolyamines, including cell membrane destabilization, endosome buffering capacity, and possibly nuclear tropism. Most importantly, addition of lipids with a triantennary galactosyl residue drives the neutral nucleolipidic particles to the asialoglycoprotein receptor of human hepatoma HepG2 cells: Transfection increases approximately 1000-fold with 25% galactolipid. This receptor-mediated process is saturable and slightly less efficient than receptor-independent transfection obtained in vitro with a large excess of cationic lipid alone. Yet, electrically silent particles may provide an attractive solution for gene transfer in vivo where their external saccharide coat should allow them to diffuse within the organism and reach their target cells.

Source of nuclear calcium signals.

Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.

A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking

The molecular mechanisms underlying the nuclear entry of steroid receptors and possible regulation of steroid hormone action during receptor passage across the nuclear envelope have not been elucidated. A nuclear localization signals has been identified in the hinge region of the glucocorticoid receptor. A synthetic peptide corresponding tothis sequence was radio-iodinated and incubated with high salt- and detergent-extracted rat liver nuclei or nuclear envelope in the presence of crosslinker. After SDS-PAGE, two nuclear polypeptides of 60 and 76 kDa which had been specifically crosslinked were identified by autoradiography. A 60 kDa polypeptide was also crosslinked in the nuclear peptides. Finally, we showed that the pattern of crosslinking of the simian virus 40 large tumour antigen nuclear localization signal was identical to that of the glucocorticoid receptor signal to the nuclear polypeptides. The crosslinked peptides are good candidates for nuclear importers of the glucocorticoid receptor. In addition, the data suggest that these binding sites may be part of a general mechanism for nuclear entry of proteins.

A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans

We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli β-galactosidase (βGal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5′ and 3′ ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3′ regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to βGal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional βGal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the βGal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.

Yeast proteins that recognize nuclear localization sequences.

A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Ga14 and histone H2B. These proteins are assayed by the binding of peptide-albumin conjugates to proteins immobilized on nitrocellulose filters. These binding proteins fractionate with nuclei and are extractable with salt but not detergent. Radiolabeled peptide-albumin conjugates also bind to isolated nuclei; the binding is saturable and can be extracted with salt. Different nuclear localization peptides compete with each other, implying that a single class of proteins is responsible for their recognition. The 70- and 59-kD proteins have the properties expected for a receptor that would act to direct proteins to the nucleus.