Biomedical subjects

X Zeng

Publications and source records attributed to X Zeng.

At least 19 recordsLinked to original sources

PubMed2001

Akt1 induces extracellular matrix invasion and matrix metalloproteinase-2 activity in mouse mammary epithelial cells.

The roles of the protein-serine/threonine kinase, Akt1, in signaling pathways associated with cell motility and extracellular matrix invasion were examined in the immortalized mouse mammary epithelial cell line, COMMA-1D. COMMA-1D cells were engineered to express the avian leukosis subtype A receptor, tv-a, to permit infection by recombinant avian leukosis virus produced by the replication-competent avian splice vector, RCAS. COMMA-1D/tv-a cells transduced with RCAS/v-akt, but not RCAS/Akt1, formed anchorage-independent colonies in soft agar; however, cells overexpressing either v-akt or Akt1 became highly invasive when grown on the ECM, Matrigel. Zymography of extracellular protease activity shed into the medium by COMMA-1D/Akt1 or COMMA-1D/v-akt cells revealed elevated gelatinase activity that was confirmed to be matrix metalloproteinase-2 (MMP-2; gelatinase A) by Western blotting and immunoprecipitation-zymography. The MMP inhibitor, BB-94, blocked MMP-2 activity and invasion associated with Akt1- and v-akt-expressing cells. The proteasome inhibitor, lactacystin, markedly increased MMP-2 levels and invasion in control cells but not in Akt1- and v-akt-expressing cells. These results suggest that the invasive behavior of mammary epithelial cells induced by Akt1 is associated with increased MMP-2 expression that may result from inhibition of MMP-2 degradation by the proteasome pathway.

Animals↗

PubMed2001

A freely diffusible form of Sonic hedgehog mediates long-range signalling.

The secreted protein Sonic hedgehog (Shh) exerts many of its patterning effects through a combination of short- and long-range signalling. Three distinct mechanisms, which are not necessarily mutually exclusive, have been proposed to account for the long-range effects of Shh: simple diffusion of Shh, a relay mechanism in which Shh activates secondary signals, and direct delivery of Shh through cytoplasmic extensions, termed cytonemes. Although there is much data (using soluble recombinant Shh (ShhN)) to support the simple diffusion model of long-range Shh signalling, there has been little evidence to date for a native form of Shh that is freely diffusible and not membrane-associated. Here we provide evidence for a freely diffusible form of Shh (s-ShhNp) that is cholesterol modified, multimeric and biologically potent. We further demonstrate that the availability of s-ShhNp is regulated by two functional antagonists of the Shh pathway, Patched (Ptc) and Hedgehog-interacting protein (Hip). Finally, we show a gradient of s-ShhNp across the anterior-posterior axis of the chick limb, demonstrating the physiological relevance of s-ShhNp.

Animals↗

PubMed2001

Altered nucleotide misinsertion fidelity associated with poliota-dependent replication at the end of a DNA template.

A hallmark of human DNA polymerase iota (poliota) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single-stranded template. Here, we report on the efficiency and accuracy of poliota-dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Poliota cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, poliota's ability to blunt-end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8- and 3-fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that poliota can extend all 12 mispairs, but with differing efficiencies. Poliota can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of poliota appear consistent with that of a somatic hypermutase and suggest that poliota may be one of the low-fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.

Animals↗

PubMed2001

The TCR repertoire of an immunodominant CD8+ T lymphocyte population.

The TCR repertoire of an epitope-specific CD8(+) T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8(+) T cell population that recognizes a dominant epitope of the AIDS virus, the CD8(+) T cells recognizing the tetrameric Mamu-A*01/p11C(,CM) complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01(+) rhesus monkeys. This CD8(+) T cell population exhibited selected usage of TCR V beta families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8(+) T cell response was clearly polyclonal, a dominance of selected V beta(+) cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected V beta(+) cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other V beta(+) cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected V beta(+) cell subpopulations and clones and suggests that dominant V beta(+) cell subpopulations and clones can either be stable or evolve during a chronic infection.

Amino Acid Sequence↗

PubMed2001

Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia.

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice approximately 100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-gamma and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-gamma and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-gamma drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.

Animals↗

PubMed2001

p300 does not require its acetylase activity to stimulate p73 function.

We previously reported that p73, like p53, utilizes p300 or cAMP-response element-binding protein-binding protein as its coactivator. Here, we extended this work by further examining whether the intrinsic acetylase activity of p300 is necessary for stimulating p73 function. Although p300 acetylated the C-terminal fragment of p73 (amino acids 311-636) in vitro, it was unable to efficiently acetylate the full-length p73. Consistently, p300 did not acetylate p73 in vivo when both the proteins were overexpressed in cells. Also, an acetylase-defective mutant p300 named p300AT2 was able to elevate p73-dependent transcription in cells. p300 associated with p73 when forming DNA-protein complexes and stabilized p73 proteins. These results demonstrate that p300 does not need its acetylase activity to be a coactivator of p73.

Acetylation↗

PubMed2001

TGF-beta1 perturbs vascular development and inhibits epithelial differentiation in fetal lung in vivo.

Members of the transforming growth factor beta (TGF-beta) family of polypeptides have been implicated in morphogenesis and differentiation in numerous tissues, including the lung. In order to further define effects of TGF-beta signaling in lung morphogenesis, a constitutively active form of TGF-beta1 was expressed in respiratory epithelial cells of the fetal mouse lung in vivo. Expression of TGF-beta1 arrested lung morphogenesis in the pseudoglandular stage of development, inhibiting synthesis of differentiation-dependent proteins, SP-B, SP-C, and CCSP, and maintaining embryonic patterns of staining for thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3beta (HNF-3beta). The pulmonary mesenchyme was thickened and vascular density was increased by TGF-beta1. TGF-beta1 decreased expression of vascular endothelial growth factor-A (VEGF-A) mRNA and protein, and the abundance of Flk-1 mRNA in the lung mesenchyme. Distribution of platelet-endothelial cell adhesion molecule (PECAM)-1, a marker of pulmonary blood vessels, was altered, and ultrastructural studies demonstrated that TGF-beta1 inhibited vascular development in the fetal lung. TGF-beta1 perturbed both epithelial cell differentiation and formation of the pulmonary vasculature, supporting the concept that precise control of signaling via the TGF-beta receptor pathway is critical for normal lung morphogenesis.

Animals↗

PubMed2001

Atrial L-type Ca2+-channel, beta-adrenorecptor, and 5-hydroxytryptamine type 4 receptor mRNAs in human atrial fibrillation.

Molecular and electrical remodeling of ion channels determining action potential duration has been proposed as a major mechanism in chronic atrial fibrillation. We investigated the mRNA expression of the cardiac L-type Ca2+-channel subunits alpha1c, alpha2/delta1, beta1a, and beta1b/c in atrial tissue of patients with chronic atrial fibrillation compared to patients in sinus rhythm. In addition, the mRNA expression of the 5-hydroxytryptamine type 4-, beta1-, and beta2-adrenergic receptors, which are known to stimulate the L-type Ca2+-current in human atrium, was analyzed and the effect of chronic beta-blocker treatment on the mRNA expression of these receptors and of the L-type Ca2+-channel subunits was assessed. Total RNA was isolated from right atrial appendages of patients in sinus rhythm and of patients with chronic atrial fibrillation. Then, semiquantitative RT-PCR using 18S RNA as the "housekeeping gene" was performed. In patients with chronic atrial fibrillation, there were only mild reductions in mRNA expression of the alpha1c-subunit (-15.5 %, p = 0.13), and of the beta1-subunit isoforms a and c (-13.3 %, p = 0.14 and -16.6%, p = 0.18, respectively). However, mRNA expression of the alpha2/delta1-subunit (-31.5 %, p < 0.01) and of the beta1-subunit isoform b (-39.9 %, p < 0.0005) was significantly reduced in patients with chronic AF. Taken together, the mRNA expression of the beta1-subunit isoforms b and c, which are splice variants, was significantly down-regulated by 26.5 % (p < 0.05) in these patients. The analysis of the beta1c/beta1b ratio resulted in a significant shift by 39.2 % (p < 0.0001) in favor of beta1c in patients with chronic atrial fibrillation. In the AF patients, the abundance of the 5-HT4-receptor transcript was significantly reduced by 36 % (p < 0.05). The beta-adrenoreceptor transcription was unchanged. In both SR and AF patients, chronic beta-blocker treatment did neither significantly effect the mRNA expression of the L-type Ca2+-channel subunits, the beta-adrenoreceptor subtypes 1 and 2, nor that of the 5-HT4-receptor. Our data show that chronic AF is associated with a decrease in the atrial mRNA amount of auxiliary subunits of the L-type Ca2+-channel and of the 5-HT4-receptor. This supports the hypothesis that the observed alterations in mRNA transcription in AF patients may lead to a decrease in the availability of functional L-type Ca2+-channels and 5-HT4-receptors and/or reduce L-type Ca2+-current amplitude and density, thus, promoting and stabilizing the arrhythmia.

Adrenergic beta-Antagonists↗

PubMed2001

Effects of homocysteine on murine splenic B lymphocyte proliferation and its signal transduction mechanism.

OBJECTIVE: Elevated plasma homocysteine (Hcy) levels have been defined as an increased risk of atherosclerosis. However, the mechanisms that Hcy induces the development of atherosclerosis are not fully understood. Therefore, effect of Hcy on B lymphocyte proliferation and its cellular mechanism were examined in normal and hyperhomocysteinemia ApoE-knockout mice. METHODS: Mouse B lymphocytes were incubated with Hcy, related compounds and/or antioxidants and/or inhibitors of PKC, p38 MAPK, NF-kappaB in the presence or absence of lipopolysaccharide. DNA synthesis, production of reactive oxygen species was measured. RESULTS: Hcy (0.1-3.0 mM) and other compounds with thiol (-SH), such as cysteine and glutathione significantly increased resting and lipopolysaccharide-induced B lymphocyte proliferation. ApoE-knockout mice with hypercysteinemia (plasma Hcy levels were 20.3+/-2.9 vs. 2.6+/-0.6 microM in control, P<0.05) had a significant promotion of B cell proliferation in response to lipopolysaccharide. Hcy also increased intracellular reactive oxygen species production. Radical scavengers reduced Hcy-induced B lymphocyte proliferation. The promotion of Hcy was significantly inhibited by inhibitors of PKC (calphostin C and RO-31-8220), p38 MAPK (SB 202190 and PD 169316) and NF-kappaB (pyrrolidine dithiocarbamate). CONCLUSIONS: The reactive oxygen species generated by thiol (-SH) auto-oxidation of Hcy are essential, and PKC, p38 MAPK and NF-kappaB are involved in the Hcy-induced B lymphocyte proliferation. Hyperhomocysteinemia may increase B lymphocyte susceptibility to inflammatory progression of atherosclerotic lesions.

Analysis of Variance↗

PubMed2001

Dissociation and unfolding of GCN4 leucine zipper in the presence of sodium dodecyl sulfate.

The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration. Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration. However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM. The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio. Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded. The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.

Circular Dichroism↗

PubMed2001

A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1.

Phosphorylation of the human p53 protein at Ser-392 has been shown to be responsive to UV but not gamma irradiation. Here we describe identification and purification of a mammalian UV-activated protein kinase complex that phosphorylates Ser-392 of p53 in vitro. This kinase complex contains casein kinase 2 (CK2) and the chromatin transcriptional elongation factor FACT (a heterodimer of hSpt16 and SSRP1). In vitro studies show that FACT alters the specificity of CK2 in the complex such that it selectively phosphorylates p53 over other substrates including casein. In addition, phosphorylation by the kinase complex enhances p53 activity. These results thus provide a potential mechanism for p53 activation by UV irradiation.

Amino Acid Sequence↗

PubMed2001

DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes.

To determine whether DNA polymerase eta plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase eta had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase eta is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.

Animals↗

PubMed2001

NBP is the p53 homolog p63.

We previously identified a non-p53, p53-responsive DNA element (p53RE)-binding protein named NBP, functionally analogous to p53, from human cervical carcinoma Hela cells. Here we report a biochemical study demonstrating that this activity is the recently cloned p53 analog p63. NBP was purified through conventional and DNA affinity chromatography to apparent homogeneity with a prominent polypeptide migrating in between the 43 and 68 kDa positions on a SDS gel. This polypeptide immunoreacted with monoclonal anti-p63 but not anti-p53 or anti-p73 antibodies. Also, NBP co-purified with p63 through each step of fractionation, as detected with anti-p63 antibodies. DNA-protein complexes formed with purified NBP and p53RE-containing oligomers derived from the p21(waf1) promoter were supershifted by anti-p63 but not anti-p53 antibodies. Thus, these results demonstrate that NBP is encoded by the p53 homolog p63 gene.

Blotting, Western↗

PubMed2001

Transient transgenic expression of gamma interferon promotes Legionella pneumophila clearance in immunocompetent hosts.

Gamma interferon (IFN-gamma) and T1-phenotype immune responses are important components of host defense against a variety of intracellular pathogens, including Legionella pneumophila. The benefit of intrapulmonary adenovirus-mediated IFN-gamma gene therapy was investigated in a nonlethal murine model of experimental L. pneumophila pneumonia. Intratracheal (i.t.) administration of 10(6) CFU of L. pneumophila induced the expression of T1 phenotype cytokines, such as IFN-gamma and interleukin-12 (IL-12). Natural killer cells were identified as the major cellular source of IFN-gamma. To determine if enhanced expression of IFN-gamma in the lung could promote pulmonary clearance of L. pneumophila, we i.t. administered 5 x 10(8) PFU of a recombinant adenovirus vector containing the murine IFN-gamma cDNA (AdmIFN-gamma) concomitant with L. pneumophila. We observed a 10-fold decrease in lung bacterial CFU at day 2 in the AdmIFN-gamma-treated group compared to controls (P < 0.01). Alveolar macrophages isolated from AdmIFN-gamma-treated animals displayed enhanced killing of intracellular L. pneumophila organisms ex vivo. Similar improvements in bacterial clearance were observed with i.t. recombinant IFN-gamma treatment. The transient transgenic expression of IL-12, a known inducer of IFN-gamma and promoter of T1-type immune responses, resulted in more modest improvement in bacterial clearance (sixfold reduction; P < 0.05). These results demonstrate that, even in immunocompetent hosts, exogenous administration or transient transgenic expression of IFN-gamma, and to a lesser extent IL-12, may be of potential therapeutic benefit in the treatment of patients with Legionella pneumonia.

Animals↗

PubMed2001

Alveolar macrophage deactivation in murine septic peritonitis: role of interleukin 10.

Sepsis predisposes the host to a number of infectious sequelae, particularly the development of nosocomial pneumonia. Mechanisms by which sepsis results in impairment of lung antibacterial host defense have not been well defined. Alveolar macrophages (AM) represent important immune effector cells of the lung airspace. In this study, we examined the effects of cecal ligation and puncture (CLP) on murine AM function ex vivo, including the expression of proinflammatory cytokines and AM phagocytic activity. AM were harvested from mice subjected to a sham operation and CLP 24 h after laparotomy, adherence purified, and challenged with lipopolysaccharide (LPS) or left unstimulated. Both unstimulated and LPS-stimulated AM from mice subjected to CLP (CLP mice) produced significantly smaller amounts of proinflammatory cytokines tumor necrosis factor alpha and interleukin (IL-12) and C-X-C chemokines KC and macrophage inflammatory protein 2 than similarly treated AM from animals subjected to a sham operation. Furthermore, AM isolated from CLP mice displayed a marked impairment in phagocytic activity, as determined by flow cytometry, with this defect persisting to 48 h post-CLP. Induction of peritoneal sepsis syndrome resulted in a time-dependent increase in IL-10 in plasma and peritoneal fluid. Interestingly, the impairment in AM proinflammatory-cytokine production and phagocytic activity observed in AM from CLP mice was partially reversed by the in vivo neutralization of IL-10 prior to AM harvest. These observations suggest that abdominal sepsis syndrome results in significant impairment in AM effector cell function, which is mediated, in part, by sepsis-induced expression of IL-10.

Animals↗

PubMed2001

Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: potential role of neutrophils as immunoregulatory cells.

The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophila pneumonia. After intratracheal inoculation of 10(6) CFU of L. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti-MIP-2 antibody resulted in an approximately 20% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (IL-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophila pneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine IL-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.

Animals↗

PubMed2001

TspO as a modulator of the repressor/antirepressor (PpsR/AppA) regulatory system in Rhodobacter sphaeroides 2.4.1.

The TspO outer membrane protein of Rhodobacter sphaeroides has been shown to be involved in controlling the transcription of a number of genes which encode enzymes involved in photopigment biosynthesis and the puc operon. The display of regulated genes appears identical to those genes encompassing the PpsR/AppA repressor/antirepressor regulon, although the effect of TspO is modest relative to that of PpsR/AppA. To directly address the hypothesis that TspO is effective through the PpsR/AppA system, we constructed mutant strains with mutations in both tspO and appA. In all cases, the phenotypes examined resembled those of the appA lesion by itself, leading us to conclude that TspO works through or modulates the PpsR/AppA system and acts upstream of the site of action of these regulatory proteins. In earlier publications, we had suggested that TspO is involved in the efflux of a certain intermediate(s) of the porphyrin biosynthesis pathway and that transcriptional regulation of target gene expression could be explained by the accumulation of a coactivator of AppA function. Although the data reported here do not precisely identify this coactivator, they lend support to this hypothesis. We discuss the importance of this form of gene control as the result of the recent extension of the TspO system to Sinorhizobium meliloti, as described by Davey and de Bruijn (M. E. Davey and F. J. de Bruijn, Appl. Environ. Microbiol. 66:5353-5359, 2000). It is therefore possible that this system constitutes a more widely, although not universally, demonstrated form of gene regulation.

Bacterial Proteins↗

PubMed2001

Development of a fluorescence based high throughput assay for antagonists of the human chorionic gonadotropin receptor extracellular domain: analysis of peptide inhibitors.

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

Automation↗