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PubMed1990

Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases.

The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.

Antiviral Agents↗
PubMed1989

Azidothymidine triphosphate is an inhibitor of both human immunodeficiency virus type 1 reverse transcriptase and DNA polymerase gamma.

The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.

DNA Polymerase III↗
PubMed1991

Characterization of an HIV-1 isolate displaying an apparent absence of virion-associated reverse transcriptase activity.

In characterizing a group of independent human immunodeficiency virus (HIV-1) isolates, we noted that certain isolates had anomolously low levels of virion-associated reverse transcriptase activity. In an attempt to understand the basis of this phenomenon, we examined in detail one such isolate, HIV-1G. We found correctly processed forms of the viral reverse transcriptase in virions as well as processed forms of other viral proteins, suggesting that viral proteins are both expressed and properly processed. We have detected a nuclease activity associated with the outer face of the HIV-1G envelope. This nuclease degrades the DNA product generated during the reverse transcription assay. The nuclease activity is more sensitive to mild protein denaturation than is the viral reverse transcriptase, and it is stimulated by the presence of Ca2+. The amount of virion-associated nuclease activity relative to reverse transcriptase activity varies between virus isolates and can vary also for one isolate during virus spread through a culture. The origin of the nuclease activity is unknown but is presumed to be cellular. The variability in amount of nuclease activity may reflect variability in the interaction of the virus with different cellular components during maturation.

Calcium↗
PubMed2004

Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR.

A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome.

Animals↗
PubMed2002

Quantitative structure-activity relationship study of 2-arylsulfonyl-6-substituted benzonitriles as non-nucleoside reverse transcriptase inhibitors of HIV-1.

The reverse transcriptase inhibition of HIV-1, the most common form of HIV, by non-nucleoside 2-arylsulfonyl-6-substituted benzonitriles is analysed through Fujita-Ban and Hansch approaches. The analyses have helped to ascertain the role of different substituents in explaining the observed inhibitory actions of these compounds. From both approaches it appeared that SO2 instead of SO or S at X; and NH2 instead of F at Y (see Figure 1) are advantageous to improving the activity of a compound against HIV-1. This in turn leads to the suggestion that the 2-arylsulfonyl-6-aminobenzonitrile scaffold is the only appropriate structural entity that may further result into potential compounds. Further, the compounds having a OMe substituent at the orthoposition, the bulkier substituents at meta-positions and "no" substituent at para-position of 2-arylsulfonyl moiety are beneficial in raising the activity. The two quantitative structure-activity relationship (QSAR) analyses, differing in parametric approach, therefore, provided the grounds for rationalizing the substituent selection in designing more potent compounds of the series.

Anti-HIV Agents↗
PubMed2003

Toxicity of non-nucleoside analogue reverse transcriptase inhibitors.

The non-nucleoside reverse transcriptase inhibitors (NNRTI) nevirapine (NVP), efavirenz (EFV), and delaviridine (DLV) are increasingly being used to treat HIV infection. Studies have shown excellent tolerance and efficacy and less development of virological resistance with HIV regimens that include NNRTIs. Nevertheless, abnormalities in liver enzymes are common in patients with HIV infection, and there are multiple etiologies for these abnormalities, including drug toxicity, viral hepatitis, opportunistic infections, and substance abuse. In particular, highly active antiretroviral therapy (HAART) can result in hepatotoxicity through a variety of mechanisms, such as mitochondrial toxicity, lipodystrophy syndrome, and steatohepatitis. The NNRTIs have been most frequently implicated in hypersensitivity reactions. NVP-containing HAART regimens may be more hepatotoxic than are those with EFV and DLV, at least for the first 6 weeks, although the data are still contradictory. Coinfection with hepatitis C and B viruses appears to significantly increase the risk of toxicity, and therefore all patients should be screened for viral hepatitis prior to commencing HAART. Close monitoring of transaminases is suggested in all patients commencing HAART, especially those with preexisting liver disease and coinfection with viral hepatitis.

Alkynes↗
PubMed1994

Kinetic analysis of the catalysis of strand transfer from internal regions of heteropolymeric RNA templates by human immunodeficiency virus reverse transcriptase.

The kinetic mechanism of HIV reverse transcriptase catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142 nucleotide RNA template (donor), primed with a specific 20 nucleotide DNA oligonucleotide that was used to initiate synthesis. An RNA with homology to an internal region of the donor was used as acceptor template. Using 32P-labeled DNA oligonucleotide, the primer-extension products made from full-length synthesis on the donor (108 bases in length) or homologous transfer to and extension on the acceptor (155 bases) were monitored. Results indicated that the maximum efficiency of transfer (the ratio of transfer products to donor-directed+transfer products x 100) in this particular system was about 25% while the theoretical Vmax for the rate of appearance of transfer products at infinite acceptor concentration was about 20-fold lower than the measured rate for full-length donor-directed products. The Km for acceptor template in the transfer reaction was about 8 nM. Experiments using the above donor template hybridized to a specific DNA that has been shown to transfer to the acceptor indicated that RNase H-mediated rapid release of this DNA from the donor while subsequent association with the acceptor was relatively slow.

Catalysis↗
PubMed2002

Substitutions of Phe61 located in the vicinity of template 5'-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase.

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.

Adenosine Triphosphate↗
PubMed2004

Direct, DNA pol-gamma-independent effects of nucleoside reverse transcriptase inhibitors on mitochondrial bioenergetics.

Nucleoside reverse transcriptase inhibitor (NRTI)-induced cardiomyopathy has been suggested to reflect mitochondrial targets of drug toxicity. The prevailing hypothesis is that, through structural mimicry, the NRTIs are mistaken as substrates for DNA polymerase and incorporated into replicating DNA, where they cause truncation of the elongating strand. Although there exist five forms of nuclear DNA polymerase, mitochondria possess solely DNA polymerase-gamma (pol-gamma), which is a preferred target for most NRTIs. Consequently, mitochondria are particularly susceptible to inhibition of DNA replication by the NRTIs, which is consistent with the phenotype of mitochondrial depletion and metabolic failure in affected patients. However, the DNA pol-gamma hypothesis by itself fails to explain the entire array of metabolic deficiencies associated with NRTI-induced disorders. In this article, we review the published literature regarding the direct effects of NRTIs on various mitochondrial targets and suggest the possibility that the initiating event in NRTI-induced cardiomyopathy is a direct mitochondrial toxicity rather than inhibition of mitochondrial DNA pol-gamma. The goal of this review is to encourage a discussion of the cause of NRTI-induced mitochondrial cardiomyopathy to include a fresh consideration of all possible targets and integrating pathways that are involved in establishing mitochondrial bioenergetic fidelity and metabolic capacity in the affected myocardium.

Animals↗
PubMed2004

Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation.

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag-pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.

Animals↗
PubMed2004

A role for dNTP binding of human immunodeficiency virus type 1 reverse transcriptase in viral mutagenesis.

HIV-1 reverse transcriptase (RT) is a highly error prone DNA polymerase. We assessed whether the ability of RT to bind nucleotide substrates affects viral mutagenesis. Structural modeling predicts that the V148 and Q151 residues influence the interaction between RT and the incoming dNTP. When we introduce either a V148I or Q151N mutation, RT fidelity increases 8.7- or 13-fold, respectively, as measured by the M13 lacZalpha forward mutation assay. Interestingly, pre-steady state kinetic studies demonstrated that these mutations do not alter polymerase fidelity during the first step of mutation synthesis, misincorporation. Rather, the V148I and Q151N mutations alter RT fidelity by weakening the ability of the polymerase to complete mismatch extension, the second step of mutation synthesis. While both these mutations minimally affect the binding of RT (K(D)) to a mismatched template-primer complex (T/P), these mutant RTs are significantly impaired in their ability to bind (K(d)) and chemically incorporate (k(pol)) nucleotide substrate onto a mismatched T/P. These differences in binding and catalysis translate into 24- and 15.9-fold increase in mismatch extension fidelity for the V148I and Q151N RT mutants, respectively. Finally, we employed a cell-based pseudotyped HIV-1 mutation assay to determine whether changes in these dNTP binding residues alter RT fidelity in vivo. We found that the V148I and Q151N mutant viruses had 3.8- and 5.7-fold higher fidelities than wild-type viruses, respectively, indicating that the molecular interaction between HIV-1 RT and the dNTP substrate contributes to viral mutagenesis.

Amino Acid Substitution↗
PubMed2002

Increased long-term mitochondrial toxicity in combinations of nucleoside analogue reverse-transcriptase inhibitors.

BACKGROUND: Some nucleoside analogue reverse transcriptase inhibitors (NRTI) may cause depletion of mitochondrial (mt) DNA in liver by inhibiting polymerase-gamma. mtDNA depletion may contribute to lactic acidosis, steatohepatitis and liver failure. OBJECTIVE: To evaluate the long-term mitochondrial toxicity of NRTI combinations. METHODS: The HepG2 human hepatoma cell line was cultivated in the presence of zalcitabine (ddC), didanosine (ddI), stavudine (d4T), lamivudine (3TC), zidovudine (ZDV) and efavirenz at concentrations equivalent to steady-state peak plasma levels (C ), and also in one-third and 10 times C. The NRTI were added to the medium alone or in combination. Control cells were incubated without any NRTI or with efavirenz. Cell growth, lactate production, intracellular lipid droplets, mtDNA and the mtDNA-encoded respiratory chain subunit COX II were monitored over a period of up to 30 days. RESULTS: Time- and dose-dependent mtDNA depletion was observed with ddC > ddI > d4T and mtDNA depletion preceded or coincided with a decline in COX II expression, a decrease in cell growth, increased lactate production and increased intracellular lipids. 3TC and efavirenz did not affect any measurement. ZDV increased lactate moderately and cell growth was inhibited, despite normal mtDNA and COX II levels. The negative effects on some measurements were more pronounced in the 3TC-ZDV and ddC-d4T combinations, than in the single-NRTI incubations. The combination of ddI-d4T was not more toxic than ddI alone. Mitochondrial damage by ZDV, d4T, ddI, and ddC did not reach steady-state by day 25. Using a Southern blot technique, mtDNA deletions were never observed. CONCLUSION: The data indicate additive or synergistic long-term mitochondrial toxicity in some NRTI combinations.

Acidosis, Lactic↗
PubMed1999

Detection of circulating prostate specific antigen expressing prostatic cells in the bone marrow of radical prostatectomy patients by sensitive reverse transcriptase polymerase chain reaction.

PURPOSE: The reverse transcriptase polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) expressing cells in the blood circulation has been under intense investigation since 1992. Although it has been suggested that this technology could be used as molecular staging for occult prostatic hematogenous metastases, we have been unable to confirm RT-PCR PSA positivity of peripheral blood to predict stage or recurrence in radical prostatectomy cases. We performed bone marrow RT-PCR PSA assay on a large cohort of radical prostatectomy cases and evaluate the use of this assay in improving prostate cancer staging and detecting early recurrence. MATERIALS AND METHODS: Unilateral anterior iliac crest bone marrow aspirates were performed on 116 patients immediately before radical prostatectomy between February 1995 and September 1997. Radical prostatectomy specimens were processed as whole mounts. A sensitive nested RT-PCR assay with specific primers derived from the PSA sequence was used, which enabled us to detect PSA expressing LNCaP prostate cancer cells at the sensitivity of 1 cancer cell per 10 million lymphocytes (1/10(7)). A minimum of 3 RT-PCR PSA reactions were performed on all patients and at least 2 positive tests were required to define positivity. Patients were followed for PSA recurrence (mean followup 14.7 months). RESULTS: PSA expressing cells were detected in bone marrow of 51 of 116 patients (44.0%) when at least 2 of 3 RT-PCR PSA assays per patient were positive. A much higher rate of RT-PCR PSA positivity was noted (77/116 patients, 66.3%) when any RT-PCR PSA positivity was considered. In 10 randomly selected cases the RT-PCR product was confirmed as PSA by deoxyribonucleic acid sequencing. Of 51 bone marrow RT-PCR positive cases 25 (49%) had organ confined disease and 26 (51%) had nonorgan confined disease. Similarly, bone marrow RT-PCR PSA was not associated with age, race, grade, pretreatment PSA or prostatic acid phosphatase value, clinical stage or margin status. However, the 2-year disease-free survival was 96.6% in RT-PCR negative patients versus 77.5% in RT-PCR positive patients (p = 0.054), and bone marrow RT-PCR PSA was an independent prognostic factor in multivariate analysis including PSA, Gleason grade and pathological stage. CONCLUSIONS: Bone marrow RT-PCR PSA positivity in this study did not predict pathological stage, grade or margin positivity as determined from whole mount prostate cancer specimens. Furthermore, no relationship with age, grade or serum markers and bone marrow RT-PCR PSA positivity was noted. However, bone marrow RT-PCR PSA was associated with early disease recurrence. Further studies and longer followup are warranted to define the metastatic potential of the PSA expressing cells in the bone marrow of prostate cancer patients.

Aged↗
PubMed2000

Antiviral resistance of biologic HIV-2 clones obtained from individuals on nucleoside reverse transcriptase inhibitor therapy.

OBJECTIVE: To study phenotypic and genotypic resistance of HIV-2 against nucleoside reverse transcriptase inhibitors (NRTI). METHODS: Biologic HIV-2 clones were generated from 3 patients before and after initiation of antiretroviral therapy with zidovudine (AZT) in patient RH2-7, AZT and didanosine (ddI) in patient PH2-1, and after addition of lamivudine (3TC) to AZT monotherapy in patient RH2-5. The sensitivity to NRTI of the virus clones, as defined by the 50% inhibitory concentration (IC(50)), was determined in vitro. The predicted amino acid sequences of the reverse transcriptase proteins from these clones were determined. RESULTS: Comparing the sensitivity of the biologic HIV-2 clones obtained after start of therapy with those from antiviral naive patients, resistance had developed to AZT (patients RH2-7 and RH2-5) and 3TC (patient PH2-1 and RH2-5). No resistance to AZT was observed in the biologic clone from PH2-1 obtained after start of therapy. The resistant clones from RH2-5 and PH2-1, but not RH2-7, contained amino acid mutations at positions where HIV-1 has been shown to mutate after AZT and 3TC treatment. CONCLUSIONS: Phenotypic resistance of HIV-2 to nucleoside analogues, which developed in HIV-2-infected patients treated with NRTIs, was associated with genotypic changes. Some of the mutations at amino acid positions in the HIV-2 reverse transcriptase gene corresponded with those involved in HIV-1 resistance, although no conventional mutations associated with resistance to AZT were observed.

Adult↗
PubMed1989

Inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxynucleoside triphosphates: template dependence, and combination with phosphonoformate.

The 2',3'-dideoxynucleoside triphosphates (ddNTPs) are potent substrate analog inhibitors of human immunodeficiency virus (HIV) reverse transcriptase and have clinical utility in the treatment of acquired immunodeficiency syndrome. Several issues regarding the interaction of these compounds with HIV reverse transcriptase were examined. The potency of unsubstituted ddNTPs and the 3'-azido analog of dTTP (AZTTP) was influenced by the choice of template. Both compounds were more potent with the complementary homopolymer templates than with gapped duplex DNA, although the Km for the competing dNTP was similar with different templates. The Ki for AZTTP was greater than for the unsubstituted ddNTPs with either a homopolymer or a gapped duplex DNA template. HIV reverse transcriptase incorporated ddCMP and AZTMP into primed phage m13 DNA at sites specified for insertion of dCMP and dTMP, respectively. ddCTP was more efficiently utilized as a substrate than was AZTTP. Primer elongation due to base misincorporation was observed in the absence of one dNTP. The combined effect of ddNTPs and the pyrophosphate analog phosphonoformate (PFA) on HIV reverse transcriptase was also examined, and inhibition by PFA in combination with ddTTP or AZTTP was mutually exclusive.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.

X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.

Crystallography, X-Ray↗
PubMed2003

Synthesis and anti-HIV activity of some heterodimers [NRTI]-glycyl-succinyl-[trovirdine analogue] of known HIV-1 reverse transcriptase inhibitors.

Expected for their ability to inhibit HIV replication, four heterodimers with a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and a Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) bound by a linker arm were designed and synthesized. For the NRTIs, d4U, d2U, d4T and 5'-O-acetyl-5-(3-hydroxypropynyl)d2U were chosen. For the NNRTI, a Trovirdine Analogue (belonging to the phenethylthiazolylthiourea class) was chosen. The conjugation of the two different inhibitors (NRTI and NNRTI) was performed using the succinyl-glycine moiety as a spontaneously cleavable linker.

Anti-HIV Agents↗
PubMed1975

Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay.

A radioimmunoassay was developed that can detect and quantitate 3 ng or more of the avian RNA tumor virus reverse transcriptase. The assay detected no antigenic sites in Rous sarcoma virus alpha virions or in virions of a murine RNA tumor virus. About 70 molecules of reverse transcriptase were found per virion of avian myleloblastosis virus with this assay or with an assay based on antibody inhibition of enzymatic activity. The assay detected about 270 ng of enzyme per mg of cell protein in virus-producing cells; uninfected cells had much less antigenic material but contained some determinants able to displace radioactive antigen. No additional antigenic determinants on reverse transcriptase could be detected that were not found on the separated alpha subunit of the enzyme. Although sevenfold less sensitive than enzymatic activity as a measure of reverse transcriptase, the radioimmunoassay can detect antigen using small amounts of protein and in the presence of inhibtors.

Animals↗