Search PubMed⌕ Search

Biomedical subjects

J C Wang

Publications and source records attributed to J C Wang.

At least 163 records · Page 9Linked to original sources
PubMed1999

The phosphoenolpyruvate carboxykinase gene glucocorticoid response unit: identification of the functional domains of accessory factors HNF3 beta (hepatic nuclear factor-3 beta) and HNF4 and the necessity of proper alignment of their cognate binding sites.

Complete induction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by glucocorticoids requires a complex glucocorticoid response unit (GRU). The GRU is comprised of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and four accessory factor-binding sites [AF1, AF2, AF3, and cAMP response element (CRE)] that bind distinct transcription factors. Hepatic nuclear factor 4 (HNF4) and chicken ovalbumin upstream promoter transcription factor (COUP-TF) bind to the AF1 element and account for AF1 activity. Members of the hepatic nuclear factor 3 (HNF3) family bind to the AF2 element and provide AF2 activity. In this report, we show that the functions of AF1 and AF2 are dependent on their positions in the promoter, since they cannot substitute for each other nor can they be exchanged without a reduction in the response to glucocorticoids. We also identified the domains of HNF4 and HNF3 beta that are required for the AF1 and AF2 activities, respectively. The carboxy-terminal transactivation domain of HNF4 (amino acids 128-374) confers most of the AF1 activity, while the carboxyterminal transactivation domain of HNF3 beta (amino acids 361-458) mediates AF2 activity. These domains of HNF4 and HNF3 beta appear to have distinct roles in the response to glucocorticoids, as there are unique structural requirements for each, as judged by the failure of most other classes of transactivation domains to serve as accessory factors. These results suggest that the regulation of the PEPCK gene by glucocorticoids requires specific interactions between GR, accessory factors, and coactivators, and that the transactivation domains of AF1 and AF2 are of fundamental importance in the assembly of this multiprotein complex.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1999

Site-directed mutagenesis of two zinc-binding centers of the NADH-dependent phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10.

Phenylacetaldehyde reductase (PAR) with a unique and wide substrate range from styrene-assimilating Corynebacterium sp. strain ST-10, which is a useful biocatalyst producing chiral alcohols, has been found to belong to a family of zinc-containing, long-chain alcohol dehydrogenases (ADHs) on the basis of the primary structure similarity. The enzyme contains 2 moles of zinc per mole of subunit. The amino acid residues assumed to be three catalytic and four structural zinc-binding ligands were characterized by site-directed mutagenesis, compared with other zinc-containing, long-chain ADHs. Sixteen PAR mutants gave measurable but rather low activities toward phenylacetaldehyde, n-hexyl aldehyde, and 2-heptanone, although they maintained the activities of 8 to 16% of that of wild-type PAR for an acetophenone substrate except that the D153N mutant showed quite low activity. The results suggested that the seven residues present in PAR were probably zinc-binding ligands, and mutation in these residues caused a change in activities for some substrates.

Alcohol Oxidoreductases↗
PubMed1999

[TGF-beta 1 in the experimentally induced inflammatory periodontal tissues in miniature swines].

TGF-beta 1 is a multifunctional molecule which has unique and potent effects on many target cells and tissues. TGF-beta 1 may promote inflammatory reaction by certain intercellular interaction. TGF-beta 1 at extremely low concentrations shows strong chemotatic activity for mononuclear phagocytes and stimulates bone resorption by enhancing production of PGE2. On the other hand, TGF-beta 1 plays a very important role in the regulation of extracellular matrix turnover presumably by modulating the action of other growth factors on matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) expression. TGF-beta 1 was identified intra- and extracellularly in the inflamed gingival tissues and the distribution was associated with areas of inflammation. Sixteen miniature swines were used in this experimental gingivitis/periodontitis study. The ligatures were placed in situ for periods of 3, 5, 8 and 13 weeks and peroral innoculations of Porphyromonas gingivalis/Actinomyces viscosus into the ligatures were carried out only in the experimental group. ELISA was used to measure the levels of TGF-beta 1 in gingival tissues from the experimental and control groups. Recording of the clinical periodontal parameters was performed and the proportion of black-pigmented Bacteroides in the ligature (plaques) removed immediately prior to the biopsies was recorded. The results revealed that the concentration of TGF-beta 1 of the experimental group was higher and significantly different in statistics on the period of third week than that of the control group. The concentration of TGF-beta 1 was significantly different between the third week and the thirteenth week in the experimental group, and was negatively related to the time-length of ligatures. Furthermore, the concentration of TGF-beta 1 was negatively related to the changes of the calculus index and gingival index. These data indicated that the concentration of TGF-beta 1 of gingival tissue exhibited dynamic changes associated with the progression of experimental periodontal inflammation. The levels of TGF-beta 1 in gingival tissue may be valuable in detecting the inflammatory reaction of periodontal tissues.

Animals↗
PubMed1999

Identification of a new single nucleotide substitution on the hypoxanthine-guanine phosphoribosyltransferase gene (HPRT(Tsou) from a Taiwanese aboriginal family with severe gout.

OBJECTIVE: A new single nucleotide change of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene on coding region has been identified from a Taiwanese aboriginal family with gout. The mutation was used to screen 27 members of the family, 22 Tsou, 70 Atayal, and 76 Bunun children, the elders for whom had been found to have a high prevalence of gout. METHODS: An entire peptide of HPRT coding region was directly sequenced from the cDNA of a patient with severe gout, and by using polymerase chain reaction and restriction fragment length polymorphism to screen the other participants. RESULTS: A new nucleotide change located at nucleotide 152 (G to A transition) was found that predicted an arginine to glutamine substitution at amino acid 51. This variant was named HPRT(Tsou), and was found in 3 women and 3 men among the patient's 7 siblings, 2 boys and 2 girls among the 8 children of the siblings, and one female Tsou (4.5%, 1/22) and one female Atayal (1.4%, 1/70). The serum uric acid concentration among male HPRT(Tsou) carriers in the patient's family was significantly higher than among those who had at least one HPRT gene that did not have HPRT(Tsou). CONCLUSION: We found that the HPRT(Tsou) gene variant is partially responsible for the hyperuricemia in an aboriginal population in Taiwan known for a high incidence of gout.

Adolescent↗
PubMed1999

The effect of cervical plating on single-level anterior cervical discectomy and fusion.

The use of anterior plates for single-level cervical fusions is controversial. Previous studies that evaluated single and multiple-level fusions have shown increased and decreased fusion rates when cervical plates are used. The purpose of this study was to compare the clinical and radiographic success of single-level discectomy performed with and without anterior cervical plate fixation. During a 6-year period, 80 patients were surgically treated with a single-level anterior cervical discectomy. Forty-four patients had cervical plates, whereas 36 had fusions without plates (average follow-up, 2.3 years). The pseudarthrosis rates were 4.5% (2 of 44) for patients with plating and 8.3% (3 of 36) without plating. This difference was not significant (p = 0.653). There was no correlation of pseudarthrosis with sex, age, level of surgery, history of tobacco use, or the presence of previous anterior surgery. The amount of graft collapse for patients with plating was 0.75 mm compared with 1.5 mm for those without a plate (p = 0.026). The amount of kyphotic deformity of the fused segment was 1.2 degrees with plating compared with 1.9 degrees for patients without plating (p = 0.079). Ninety-one percent of the patients with plating had good or excellent results compared with 88% in the group without cervical plates, based on Odom's criteria. The addition of plate fixation for single-level anterior cervical discectomy and fusion is safe and not associated with a significant increase in complication rates. The pseudarthrosis rates are not significantly different when a cervical plate is used.

Adult↗
PubMed1998

Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases.

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.

Amniocentesis↗
PubMed1998

Centromeric DNA break in a 10;16 reciprocal translocation associated with trisomy 16 confined placental mosaicism and maternal uniparental disomy for chromosome 16.

Stable centromeric breakage in non-acrocentric chromosomes and balanced reciprocal translocation mosaicism are both rare events. We studied a family in which the mother had mosaicism for a balanced reciprocal translocation between chromosomes 10 and 16 which was associated with a break in chromosome 16 centromere alpha-satellite DNA ¿146,XX,t(10;16)(q11.2;q11.1) [29]/46,XX[25]¿. The derivative chromosome 16 contained only a very small amount of 16 alpha-satellite DNA while the derivative 10 contained all of the 10 alpha-satellite DNA as well as a large amount of the 16 alpha-satellite DNA. The same translocation was present in all cells in her son who was found prenatally to have trisomy 16 mosaicism ¿46,XY,t(10;16) (q11.2;q11.1)mat[22]/47,idem,+16[4]¿. Trisomy 16 cells were subsequently determined to be confined to the placenta. DNA polymorphism analyses in the family demonstrated maternal uniparental disomy for chromosome 16 in the diploid child. The child, at age 7 months, had minor facial anomalies similar to a previously reported case of maternal uniparental disomy for chromosome 16. In addition to illustrating several rare events, this family further demonstrated that substantial deletion of the centromeric alpha-satellite DNA does not impair centromere function and both mitotic and meiotic stability are retained in such cases.

Adult↗
PubMed1998

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli.

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.

Adenosine Triphosphate↗
PubMed1998

A biomechanical evaluation of graft loading characteristics for anterior cervical discectomy and fusion. A comparison of traditional and reverse grafting techniques.

STUDY DESIGN: A biomechanical study of graft loading characteristics for anterior cervical discectomy and fusion comparing the amount and location of transmitted forces. OBJECTIVES: To evaluate the difference between traditional iliac grafting and reverse iliac grafting used for anterior cervical discectomy and fusion in the amount and location of forces applied to the grafts. SUMMARY OF BACKGROUND DATA: Traditional fusion after anterior cervical discectomy involves placing a tricortical iliac crest strut into the disc space with the cortical portion facing anteriorly and the cancellous portion posteriorly. Recently, reverse iliac grafting has been introduced in which the cortical portion is placed in the posterior disc space and the cancellous portion in the anterior disc space. There is no biomechanical or clinical study showing an advantage of using one technique over the other. This study is the first to produce data supporting one technique as biomechanically superior. METHODS: Five fresh cadaveric cervical spines were tested using pressure-sensitive film placed between the bone graft and the vertebral endplate after an anterior discectomy was performed. A 10-pound load was applied to the cervical spine at predetermined sagittal positions. Recordings were made at neutral, 10 degrees of flexion, and 10 degrees and 20 degrees of extension after traditional and reverse iliac grafting. RESULTS: Graft forces were identical in both traditional and reverse grafting in the location and amount of force applied. Total force increased to the maximum in flexion and gradually decreased in more extended positions. The location of the forces was completely anterior with flexion, moving to the posterior portion of the graft with positions of extension. With 10 degrees of flexion, the load applied to the grafts was 20.4 N. In the neutral position, the load was 12 N. The loads decreased further with extension with forces of 11 N in 10 degrees extension, and 4 N in 20 degrees of extension. CONCLUSIONS: The optimal position of the tricortical iliac graft for an anterior cervical fusion is with the stronger cortical portion placed in the anterior disc space and the weaker cancellous portion placed in the posterior disc space. In this traditional position, the graft will best resist the loads applied to the cervical spine, preventing graft collapse.

Adult↗
PubMed1998

Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II.

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.

Binding Sites↗
PubMed1998

A comparison of magnetic resonance and computed tomographic image quality after the implantation of tantalum and titanium spinal instrumentation.

STUDY DESIGN: Tantalum- and titanium-based lumbar interbody fusion devices were implanted into two fresh human cadavers, and magnetic resonance and computed tomographic imaging were performed to evaluate adjacent spinal structures and the amount of metallic artifact. OBJECTIVE: The objective of this study was to prospectively compare the preliminary results of magnetic resonance imaging and computed tomography scanning image quality after the implantation of both titanium and tantalum spinal implants. SUMMARY OF BACKGROUND DATA: The availability of tantalum and titanium spinal implants brings theoretical magnetic resonance imaging compatibility along with several other desirable characteristics. The magnetic resonance imaging and computed tomographic imaging of tantalum spinal instrumentation has never been studied previously or compared with titanium instrumentation. METHODS: Titanium and tantalum spinal implants produced for anterior spinal fusion were each placed at two levels in the lumbar spine of two fresh cadaver specimens. Sequential spin echo T1-weighted and T2-weighted magnetic resonance imaging studies and computed tomographic scans were obtained. The resulting images were then graded to describe and compare the behavior of tantalum metal in magnetic resonance imaging and computed tomographic studies. RESULTS: Good T1 and T2 images were obtained that allowed visualization of the neural structures with minimal artifact. The optimal T1 images for tantalum metal were similar in quality to the optimal T1 parameters for titanium metal. T2 images for both tantalum and titanium metal were obtained with similar results for both metals. Gradient echo magnetic resonance imaging scans of both were poorly imaged with a large amount of artifact. Computed tomographic studies of tantalum implants produced a large amount of metal artifact when compared with computed tomographic studies of titanium implants. CONCLUSIONS: High-quality magnetic resonance imaging studies can be obtained after the implantation of both titanium and tantalum spinal instrumentation. Both of the metals produce similar images on magnetic resonance imaging studies with comparable amounts of metallic artifact. High-quality computed tomographic scans of titanium implants can be obtained with minimal distortion secondary to artifact. However, computed tomographic scanning is not the imaging modality of choice for the tantalum spinal implants because of the large amounts of artifact.

Artifacts↗
PubMed1998

Structural similarities between topoisomerases that cleave one or both DNA strands.

Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex. Both types have been proposed to use an "enzyme-bridging" mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment. Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA.

Binding Sites↗
PubMed1998

Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity. The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA. In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed. Similar to the E. coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E. coli single-stranded DNA-binding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

Adenosine Triphosphatases↗
PubMed1998

Analysis of FHIT transcripts in cervical and endometrial cancers.

Carcinoma of the uterine cervix is a common malignancy, and many affected women, have been found to exhibit loss of heterozygosity (LOH) in the chromosome 3p region. Recent studies have localized the FHIT (fragile histidine triad) gene in this region and also demonstrated a high frequency of abnormalities of this gene in various cancers. To determine the role of the FHIT gene in cervical and uterine carcinomas, 16 cases of cervical carcinoma and 7 cases of endometrial carcinoma, as well as nearby non-cancerous tissues in these patients, were analyzed by reverse transcription of the FHIT mRNA followed by polymerase chain reaction amplification and sequencing of the products. In this study, 13 of 16 cervical cancers and 4 of 7 endometrial cancers displayed abnormal FHIT transcripts, including a lack of 2 or more exons of the FHIT gene, the insertion of several bases in the deletion junctions, and a 282 bp deletion from cDNA 171 to 452, resulting in a frameshift. Moreover, 5 of 16 matched non-cancerous tissues from the cervical cancer patients and 4 of 7 non-cancerous tissues from endometrial cancer patients also showed the presence of abnormal transcripts lacking 3 or more exons of the FHIT gene. Only 1 of 23 paired samples exhibited LOH. Our results suggest that the abnormal transcript of the FHIT gene is common in both normal and tumor tissues of the uterus and cervix. We also checked for HPV infection in these samples and found no definite relationship between the abnormal transcript and human papillomavirus infection.

Acid Anhydride Hydrolases↗
PubMed1998

High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase.

We have previously shown that intravenously injected peripheral blood (PB) or bone marrow (BM) cells from newly diagnosed chronic myeloid leukemia (CML) patients can engraft the BM of sublethally irradiated severe combined immunodeficient (SCID) mice. We now report engraftment results for chronic phase CML cells in nonobese diabetic (NOD)/SCID recipients which show the superiority of this latter model. Transplantation of NOD/SCID mice with 7 to 10 x 10(7) patient PB or BM cells resulted in the continuing presence of human cells in the BM of the mice for up to 7 months, and primitive human CD34+ cells, including those detectable as colony-forming cells (CFC), as long-term culture-initiating cells, or by their coexpression of Thy-1, were found in a higher proportion of the NOD/SCID recipients analyzed, and at higher levels than were seen previously in SCID recipients. The human CFC and total human cells present in the BM of the NOD/SCID mice transplanted with CML cells also contained higher proportions of leukemic cells than were obtained in the SCID model, and NOD/SCID mice could be repopulated with transplants of enriched CD34+ cells from patients with CML. These results suggest that the NOD/SCID mouse may allow greater engraftment and amplification of both normal and leukemic (Ph+) cells sufficient for the quantitation and characterization of the normal and leukemic stem cells present in patients with CML. In addition, this model should make practical the investigation of mechanisms underlying progression of the disease and the development of more effective in vivo therapies.

Adult↗
PubMed1998

Identification of active site residues in Escherichia coli DNA topoisomerase I.

Alanine substitution mutagenesis of Escherichia coli DNA topoisomerase I, a member of the type IA subfamily of DNA topoisomerases, was carried out to identify amino acid side chains that are involved in transesterification between DNA and the active site tyrosine Tyr-319 of the enzyme. Twelve polar residues that are highly conserved among the type IA enzymes, Glu-9, His-33, Asp-111, Glu-115, Gln-309, Glu-313, Thr-318, Arg-321, Thr-322, Asp-323, His-365, and Thr-496, were selected for alanine substitution. Each of the mutant enzymes was overexpressed, purified, and characterized. Surprisingly, only substitution at Glu-9 and Arg-321 was found to reduce the DNA relaxation activity of the enzyme to an insignificant level. The R321A mutant enzyme, but not the E9A mutant enzyme, was found to retain a reduced level of DNA cleavage activity. Two additional mutant enzymes R321K and E9Q were also constructed and purified. Replacing Arg-321 by lysine has little effect on enzymatic activities; replacing Glu-9 by glutamine greatly reduces the supercoil removal activity but not the DNA cleavage and rejoining activities. From these results and the locations of the amino acids in the crystal structure of the enzyme, it appears that Glu-9 has a critical role in DNA breakage and rejoining, probably through its interaction with the 3' deoxyribosyl oxygen. The positively charged Arg-321 may also participate in these reactions by interacting with the scissile DNA phosphate as a monodentate. Because of the strict conservation of these residues, the findings for the E. coli enzyme are likely to apply to all type IA DNA topoisomerases.

Amino Acid Sequence↗