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PubMed1991

Sequence and linkage analysis of the Coxiella burnetii citrate synthase-encoding gene.

The nucleotide (nt) sequence of the Coxiella burnetii citrate synthase-encoding gene (gltA), previously cloned in Escherichia coli, was determined. The nt sequence analysis revealed an open reading frame (ORF) of 1290 bp capable of coding for a protein of 430 amino acids (aa) with a deduced Mr of 48,633. Preceding an ATG start codon, a possible transcription start point (tsp) with homology to the E. coli promoter consensus was detected. A poly-purine-rich region occurred immediately upstream from the gltA reading frame and potentially serves as a ribosome-binding site. Additionally, a G + C-rich region of dyad symmetry 3' to the translational stop codon was found that could possibly function as a Rho-independent transcriptional termination signal. A large, nearly perfect, inverted repeat was identified upstream from the gltA tsp and was shown by Southern analysis to be present in multiple copies in the C. burnetii genome. The deduced aa sequence of C. burnetii GltA was optimally aligned with enzymes from various prokaryotic sources and one eukaryotic source (pig heart). Using perfect aa identity, the C. burnetii enzyme demonstrated the greatest homology with GltA from Acinetobacter anitratum (65%). Although only 26% aa identity was seen with the pig heart enzyme, many of the residues identified in ligand binding appear to be conserved. Sequencing studies of a region centered approx. 5.6 kb upstream from gltA revealed an ORF read with opposite polarity that encodes a peptide highly homologous to the C terminus of the flavoprotein subunit of E. coli succinate dehydrogenase. This report represents the first nt sequence analysis of a gene of known function from the obligate intracellular parasite, C. burnetii.

Amino Acid Sequence↗
PubMed1991

Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response.

The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.

Base Sequence↗
PubMed2004

A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1.

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.

Aeropyrum↗
PubMed2003

Development of a 5' nuclease assay to detect ciprofloxacin resistant isolates of the biowarfare agent Yersinia pestis.

The detection of antibiotic resistance in agents of biowarfare is extremely critical in order to start appropriate therapy in a timely manner. We have developed a 5' nuclease assay to detect ciprofloxacin resistance (Cip(r)) in Yersinia pestis. Two groups of fluorogenic probes were developed. The first group included a probe homologous to the wild type Y. pestis gyrA sequence with two corresponding probes that were homologous with two different mis-sense mutations in codon 81 of GyrA. The second group of probes included a wild type probe and two corresponding probes that recognized mis-sense mutations in codon 83 or gyrA. These probes specifically reacted only with the homologous DNA sequences. The 5' nuclease assay was sensitive to 1 pg (approximately 1 colony forming unit) of starting template and could be used on semi-purified DNA. We tested our optimized assay against a group of 38 Cip(r) Y. pestis isolates and demonstrated that it was accurate at determining the gyrA allele encoded by these strains. The 5' nuclease assay performed on the LightCycler proved to be sensitive, rapid and accurate at identification of Cip(r) Y. pestis and thus should be useful in characterization of this organism in the event of a future act of bioterrorism.

Animals↗
PubMed1996

A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli.

We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC. Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo.

Bacterial Proteins↗
PubMed2005

In vitro activity of structurally diverse nucleoside analogs against human immunodeficiency virus type 1 with the K65R mutation in reverse transcriptase.

Human immunodeficiency virus type 1 (HIV-1) with a lysine-to-arginine substitution at codon 65 (HIV-1(65R)) of reverse transcriptase (RT) can rapidly emerge in patients being treated with specific combinations of nucleoside analog RT inhibitors (NRTIs). A better understanding of the activity of approved and investigational NRTIs against HIV-1(65R) is needed to select optimal therapy for patients infected with this mutant and to devise strategies to prevent its emergence. Therefore, we tested a broad panel of NRTIs that differed by enantiomer, pseudosugar, and base component against HIV-1(65R) to determine how NRTI structure affects activity. Drug susceptibilities of recombinant wild-type (HIV-1(65K)) or mutant HIV-1(65R) were determined using a single-replication-cycle susceptibility assay with P4/R5 cells and/or a multiple-replication-cycle susceptibility assay with MT-2 cells. All D, L, and acyclic NRTIs were significantly less active against HIV-1(65R) than against HIV-1(65K) except for analogs containing a 3'-azido moiety. Pseudosugar structure and base component but not enantiomer influenced NRTI activity against HIV-1(65R). These findings support the inclusion of 3'-azido-3'-deoxythymidine in drug combinations to treat patients having HIV-1(65R) and to prevent its emergence.

HIV Reverse Transcriptase↗
PubMed1980

Early assignments of the genetic code dependent upon protein structure.

Orgel (1972) has suggested that polynucleotides with sequences of alternating purine/pyrimidine are likely to have predominated in prebiotic conditions. Therefore, in any early template-directed protein synthesis, the number of available codons would have been limited. However, for any self-organizing system to survive and propagate, some feedback must occur from the products of the synthesis to the control of the synthetic procedure itself; i.e. the protein synthesized should have catalysed some step in the initation of template-directed synthesis. A given protein structure with a characteristic conformation and function would be optimal for the product of such a synthesis, and this in turn would limit the number of nucleotide sequences of those available able to give rise to a functioning synthetic assembly. A possible candidate for such an early polypeptide is the ferredoxin group of proteins and it is shown that with the present-day code the corresponding nucleotides do have a high percentage of alternating purine/pyrimidine sequences. Hence these combined restraints on the primitive synthetic machinery would direct the possible assignments of the genetic code helping to explain its regularity and universality.

Amino Acid Sequence↗
PubMed2000

Optimized multiplex IgH/ras PCR: a tool for quantitative monitoring of B-lymphoproliferative disorders.

The use of quantitative PCR is recommended to monitor the level of residual hematological malignancies. The proposed multiplex IgH/ras PCR uses a co-amplification of the clonal CDR3 rearrangement of the immunoglobulin heavy chain gene (IgH) as a disease marker and a segment of the Hras 1 gene containing codon 61 (ras) as a control gene. Serial dilutions of stored diagnostic DNAs are examined together in the same PCR at a sub-plateau phase and, after analysis by densitometry, the amount of CDR3 product is related to the ras product. An increase of this ratio at comparable amounts of DNA is viewed as an increase of malignant cells. This endpoint PCR quantifying approach appears to be applicable in monitoring B-lymphoproliferative disorders as was shown to be true in B-cell non-Hodgkin's lymphoma and may provide information on disease activity and treatment outcome.

B-Lymphocytes↗
PubMed1998

A New Strategy for Optimizing the Translation Initiation Rate of Foreign Gene Expression in E. coli.

The translation initiation rate is greatly affected by the secondary structure of the translation initiation region (TIR) of mRNA. A novel system was established for improving the translation initiation rate of a foreign gene in E. coli. As a model, the 5' 114 bp coding sequence (38 amino acids from the start codon) of human proliferating cell nuclear antigen (PCNA) gene was fused with the lacZ' gene at its 5' end in vector pTZ19R. A Shine/Dalgarno (SD) sequence GAGGT was inserted to the -8 position of AUG by site-directed mutation. Then the flanking sequences of SD, which were the 6 nucleotides upstream the SD and the 7 nucleotides between SD and AUG, were randomly changed by PCR using a synthetic primer with partially random sequences. This random mutation led to potential variations in the secondary structure of the TIR of mRNA through base pairing with the 5' coding sequence. The 5' PCNA-LacZ' mRNA could be efficiently and specifically transcribed by inducible T7 RNA polymerase in E. coli strain JM 109 (DE3). There were 269 clones of 5' PCNA-LacZ' fusion plasmid selected first by the blue color on X-gal plate and then by hybridization with a 5' PCNA probe. Eight clones with different blue colors among the 269 clones were chosen for beta-galactosidase activity assay. The results showed that the difference between their enzyme activities was more than 20 fold, but there seemed no apparent difference at the transcription level as assayed by RNA dot hybridization, which suggested that the difference in the expression of the fusion protein was due to the different rate of translation initiation. Thus, by this strategy, an effective translation initiation region for the high expression of human PCNA gene in E. coli was obtained.

Journal Article↗
PubMed2004

Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.

beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.

Adipates↗
PubMed2000

Increasing fidelity in parsimony analysis of dorid nudibranchs by differential weighting, or a tale of two genes.

Phylogenetic analyses of 22 dorid nudibranch species and 2 outgroup (dendronotacean and notaspidean) species were performed using sequences from two different mitochondrial genes (16S rRNA and COI). Several methods of differential weighting (positional, transformational, and combined) were explored using character congruence between the linked data sets as an optimality criterion. Most weighting schemes gave an increase in congruence as well as phylogenetic signal. The optimal weighting scheme according to the criterion was successive weighting of each character (positional weighting) with 1/(number of steps) in combination with LN weighting of character changes (transformational weighting). The cladogram from the optimal weighting scheme was, in general, congruent with existing classifications. One exception is the genus Goniodoris, which was paraphyletic if Okenia aspersa was not also included.

Animals↗
PubMed1991

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase.

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.

Acetates↗
PubMed2006

Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota).

The resolving power and statistical support provided by two protein-coding (RPB1 and RPB2) and three ribosomal RNA-coding (nucSSU, nucLSU, and mitSSU) genes individually and in various combinations were investigated based on maximum likelihood bootstrap analyses on lichen-forming fungi from the class Lecanoromycetes (Ascomycota). Our results indicate that the optimal loci (single and combined) to use for molecular systematics of lichen-forming Ascomycota are protein-coding genes (RPB1 and RPB2). RPB1 and RPB2 genes individually were phylogenetically more efficient than all two- and three-locus combinations of ribosomal loci. The 3rd codon position of each of these two loci provided the most characters in support of phylogenetic relationships within the Lecanoromycetes. Of the three ribosomal loci we used in this study, mitSSU contributed the most to phylogenetic analyses when combined with RPB1 and RPB2. Except for the mitSSU, ribosomal genes were the most difficult to recover because they often contain many introns, resulting in PCR bias toward numerous and intronless co-extracted contaminant fungi (mainly Dothideomycetes, Chaetothyriomycetes, and Sordariomycetes in the Ascomycota, and members of the Basidiomycota), which inhabit lichen thalli. Maximum likelihood analysis on the combined five-locus data set for 82 members of the Lecanoromycetes provided a well resolved and well supported tree compared to existing phylogenies. We confirmed the monophyly of three recognized subclasses in the Lecanoromycetes, the Acarosporomycetidae, Ostropomycetidae, and Lecanoromycetideae; the latter delimited as monophyletic for the first time, with the exclusion of the family Umbilicariaceae and Hypocenomyce scalaris. The genus Candelariella (formerly in the Candelariaceae, currently a member of the Lecanoraceae) represents the first evolutionary split within the Lecanoromycetes, before the divergence of the Acarosporomycetidae. This study provides a foundation necessary to guide the selection of loci for future multilocus phylogenetic studies on lichen-forming and allied ascomycetes.

Ascomycota↗
PubMed2006

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging.

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

Animals↗
PubMed2002

Detection of ethambutol-resistant Mycobacterium tuberculosis strains by multiplex allele-specific PCR assay targeting embB306 mutations.

We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.

Alleles↗
PubMed2001

Reverse transcription slippage over the mRNA secondary structure of the LIP1 gene.

The secondary structures in mRNA often cause early termination during the synthesis of cDNA. In an attempt to determine the 5'-untranslated region (UTR) of the gene LIP1 using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we found that reverse transcriptases skipped over the LIP1 RNA secondary structures and continued the DNA synthesis through RNA adapter sequences without early termination. A fragment of only three nucleotides upstream of the LIP1 translation initiation codon was obtained from the initial RACE-PCR, which was much shorter than the 57-nucleotide fragment obtained from the cDNA library screening. Analysis of the 5' end sequence indicates the presence of high G+C content and stem-loop secondary structures. Therefore, optimizations of the reaction with high temperature (70 degrees C) and a thermostable reverse transcriptase were performed to synthesize the first-strand cDNA, which was determined to have 73 nucleotides in the 5'-UTR. These results suggest that, under cDNA synthesis conditions at 42 degrees C and 60 degrees C, the reverse transcriptases skipped over the stem-loop structures of LIP1 mRNA and continued the cDNA synthesis until they reached the RNA adapter sequences. Thisfinding draws attention to adopting optimized conditions for cDNA synthesis on G+C-rich RNA templates.

5' Untranslated Regions↗
PubMed2001

Base-pair alterations in the epsilon-lower stem due to a novel double substitution in the precore gene of HBV-e negative variant were recovered by secondary mutations.

The HBe negative phenotype, a natural precore mutant (G1896A/G1897A) of HBV with aborted HBeAg expression is known to cause chronic hepatitis. The destabilized C : G base-pairing in the lower stem of epsilon-hairpin due to G1896A substitution is reportedly compensated by a second C1858T mutation and suggested to play an important role in enhanced selection of the HBe negative variant. We undertook to investigate presence of such compensatory mutations at other positions by analyzing epsilon-sequences (nts. 1847-1907) as well as to look for their effect(s), if any, on the consensus sequence of the overlapping core-initiator of HBe negative HBV variants in CLD patients. Three of the 5 HBe negative patients had classical G1896A mutation having a second compensatory mutation at nt. 1858. One patient showed an additional G1897A substitution, presenting as a novel precore stop codon mutation (UGG-->UAA), followed by a compensatory mutation at position 1857. In the third patient, a G1899A substitution was seen which compensated the impaired U at position 1855. Other substitution and deletion mutations were also observed in the remaining epsilon-hairpin, which however, did not produce any compensatory mutation. Further, all the precore variants showed a conserved G at position 1904, important for the optimal context of their core-initiator which however, remained impaired with A (nt. 1850). Our results suggest that the nts. 1851-1859 and nts. 1895-1904 in the lower stem, and restoration of authentic base-pairings therein, maintain the structural integrity and stability of the epsilon-hairpin. This may have a role in the enhanced selection of the HBe negative variants and persistence of HBV infection in chronic liver disease patients.

Base Pairing↗
PubMed1996

Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR.

Mutant-enriched PCR was applied to the detection of mutations at codons 12 and 13 of K-ras genes in the stools of patients with colorectal cancer. Mutations were analyzed in stool samples obtained prior to surgery. Resected tumor specimens were screened for K-ras mutations by PCR-mediated RFLP analysis. Using normal stool samples, assay conditions were adjusted to optimal sensitivity and specificity. The following specimens were included in the study: 16 stool samples corresponding to carcinomas in which K-ras mutations had been identified; 7 randomly selected stool samples corresponding to carcinomas which were negative for K-ras mutations; 1 stool sample from a patient with non-Hodgkin's lymphoma. In 13 of the 16 stool samples (81%) corresponding to tumors in which K-ras mutations had been identified previously, K-ras mutations were detected. In 2 of the 7 stool samples corresponding to tumors in which K-ras mutations had not been detected by previous PCR-mediated RFLP analysis, K-ras mutations were also present. Reanalyses of the tumors corresponding to these 2 positive stool samples by mutant-enriched PCR revealed a K-ras mutation in one of the tumors. The stool and tumor of the patient with non-Hodgkins lymphoma were negative for K-ras mutations. DNA sequence analysis revealed that, for each of the K-ras mutations identified in stool samples, identical base substitutions were present in the corresponding tumor tissue. The results indicate that tumor cells harboring K-ras mutations can be detected in the stools of patients with colorectal cancer by mutant-enriched PCR with high sensitivity and specificity. Because of the simplicity of the technique, it may be suitable for screening of stool samples for mutations of the K-ras gene.

Adult↗