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E M Peterson

Publications and source records attributed to E M Peterson.

At least 19 recordsLinked to original sources
PubMed1995

Value of terminal subcultures for blood cultures monitored by BACTEC 9240.

Blood cultures collected in BACTEC Plus Aerobic/F bottles and BACTEC Plus Anaerobic/F bottles were monitored for 5 days by BACTEC 9240 and subsequent terminal subcultures. Of the 13,471 bottles subcultured, 11.0% (1,477 of 13,471) were culture positive. Of these, 94.0% (1,388 of 1,477) were detected by BACTEC 9240; the additional 6.0% (89 of 1,477) were considered to be false negatives by BACTEC 9240 since they were detected by terminal subculture only. The false-negative bottles consisted of 17 BACTEC Plus Aerobic/F and 72 BACTEC Plus Anaerobic/F bottles, accounting for 2.2 (17 of 786) and 10.4% (72 of 691) of the total positive aerobic and anaerobic bottles, respectively. The positive blood culture bottles most frequently not detected by BACTEC 9240 grew Pseudomonas spp. (24), Staphylococcus spp. (21), and yeasts (24). Of the 86 blood cultures represented by the 89 false-negative bottles, 41 would not have been identified as positive since the other bottle in the blood culture set was either a false negative or a true negative. In general, terminal subcultures of false-negative BACTEC bottles had heavy growth, indicating that BACTEC Plus media were able to support the growth of microorganisms, but the BACTEC 9240 instrument was unable to detect this growth.

Adult↗
PubMed1994

Alpha-spirocyclopentyl- and alpha-spirocyclopropyl-gamma-butyrolactones: conformationally constrained derivatives of anticonvulsant and convulsant alpha,alpha-disubstituted gamma-butyrolactones.

To further study the putative gamma-butyrolactone site of the GABAA/chloride channel complex, constrained derivatives of convulsant and anticonvulsant alpha,alpha-disubstituted gamma-butyrolactones (alpha-spirocyclopropyl- and alpha-spirocyclopentyl-gamma-butyrolactones) were synthesized and evaluated biologically. Most of the spirocyclopropyl agents were anticonvulsants when tested against pentylenetetrazole-induced seizures in mice. These agents effectively displaced 35[S]-tert-butylbicyclophosphorothionate (35[S]-TBPS), a ligand for the picrotoxin binding site of the GABAA/chloride channel, from rat neuronal membranes and affected the GABA-mediated current in hippocampal neurons. The monomethyl-substituted spirocyclopropyl agent with a methyl group cis to the carbonyl (15) potentiates GABA-induced current whereas the trans derivative (16) blocks the current. The only anticonvulsant in the spirocyclopentyl series was the unsubstituted spirocyclopentyl compound 2. All the other substituted spirocyclopentyl targets were inactive in vivo at the highest dose tested except for convulsant 9, which has a trans 2,5-dimethyl-substituted cyclopentyl ring. All the spirocyclopentyl derivatives displaced 35[S]-TBPS from rat neuronal membranes very effectively, and they also all potentiated GABA-induced chloride current except for convulsant 9 which blocked the current. From the data obtained in this investigation, it appears that when the volume occupied above and below the lactone ring is as large as that occupied by spirocyclopentyl agent 9, convulsant activity is observed. Groups with less volume in these areas either are inactive in the behavioral test or have anticonvulsant activity. When bound to the GABAA/chloride channel, the larger molecules may stabilize the closed state of the channel whereas the smaller molecules may stabilize the open state.

4-Butyrolactone↗
PubMed1994

Analysis of the humoral response elicited in mice by a chimeric peptide representing variable segments I and IV of the major outer membrane protein of Chlamydia trachomatis.

A synthetic chimeric peptide representing the variable segments I (VS I) and IV of the major outer membrane protein (MOMP) of Chlamydia trachomatis, serovars C and E respectively, was studied to determine its ability to elicit a neutralizing humoral response in mice. Antibody responses varied to the peptide in the five inbred strains of mice, A/J(H-2a), DBA/1(H-2q), C57BL/10(H-2b), CBA/J(H-2k), Balb/c(H-2d), that were immunized. There was a spectrum of antibody responses which ranged from high ELISA and IFA titres by the C57BL/10 mice to little or no response by Balb/c mice. Antisera from C57BL/10 mice recognized all 15 serovars of C. trachomatis in a dot blot assay. A pepscan of the antisera from C57BL/10 mice showed strong reactivity to both neutralizing epitopes VAGLQNDPT in VS I of serovar C and the species-conserved peptide, TLNPTIA, in the VS IV. This antiserum neutralized, in vitro, the infectivity of serovars representing the B complex (B, D and E), C complex (C and J), B-related (F) and C-related (L3) complexes. In an attempt to elicit a stronger response to the peptide in the weakly reactive Balb/c and the DBA/1 strains, the peptide was conjugated to the carrier, keyhole limpet haemocyanin (KLH). All mice immunized with the KLH-peptide produced high-titred antisera that recognized neutralizing epitopes in VS I and VS IV and strongly neutralized the infectivity of both serovars C and E.

Amino Acid Sequence↗
PubMed1994

Comparison of the ProSpecT and Color Vue enzyme-linked immunoassays for the detection of Cryptosporidium in stool specimens.

Two enzyme-linked immunosorbent assays, the ProSpecT (Al-exon, Sunnyvale, CA, USA) and the Color Vue (Seradyn, Indianapolis, IN, USA) were compared for their ability to detect Cryptosporidium in 236 formalin-fixed stool specimens using the Merifluor C/G (Meridian, Cincinnati, OH, USA) stain as the reference method. The initial sensitivities of the ProSpecT and the Color Vue were 96.0% and 76.0%, which upon repeat testing of all discrepancies remained at 96.0% for the ProSpecT and decreased to 72.0% for the Color Vue. There were 25 (11%) specimens positive by the reference method. Initially, there were five false positive specimens by the ProSpecT, only one of which remained positive on retesting. The specificity of the Color Vue was 100% for the initial and repeated results, whereas the ProspecT had an initial specificity of 97.6% that increased to 99.5% upon repeat testing. The enzyme-linked immunosorbent assays offered the advantages of objectivity, batch testing, and, in the case of the ProSpecT, an acceptable sensitivity.

Animals↗
PubMed1994

Intravaginal inoculation of mice with the Chlamydia trachomatis mouse pneumonitis biovar results in infertility.

In an attempt to establish a model of chlamydial ascending salpingitis and infertility, three inbred strains of mice, C3H/HeN (H-2k), C57BL/6N (H-2b), and BALB/cAnN (H-2d), were inoculated intravaginally with 3 x 10(7) inclusion-forming units of the Chlamydia trachomatis mouse pneumonitis biovar. Mice mated 6 weeks following inoculation were found to have a significant decrease in fertility rate compared with the control groups, as shown by a reduction in the number of pregnant mice and a decrease in the number of embryos.

Animals↗
PubMed1994

Protection against infertility in a BALB/c mouse salpingitis model by intranasal immunization with the mouse pneumonitis biovar of Chlamydia trachomatis.

Female BALB/c mice were immunized intranasally with the mouse pneumonitis biovar of Chlamydia trachomatis and subsequently challenged in the ovarian bursa (C. trachomatis immunized, C. trachomatis challenged). Two groups of mice served as controls. One group was sham immunized intranasally with mock-infected HeLa 229 cell extracts and was challenged in the ovarian bursa with C. trachomatis MoPn (sham immunized, C. trachomatis challenged). The second control group was sham immunized and not challenged (sham immunized, nonchallenged). Before challenge, the C. trachomatis-immunized, C. trachomatis-challenged animals mounted a significant humoral response as shown by high immunoglobulin G (IgG), IgM, and IgA levels and high levels of neutralizing antibodies in serum and moderate IgG and IgA titers in vaginal secretions. Reactivity by Western blot (immunoblot) to the lipopolysaccharide, 30-, 40- (major outer membrane protein), and 60-kDa cysteine-rich proteins and 75- and 100-kDa chlamydial components could be demonstrated. However, reactivity to the 60-kDa heat shock protein was only observed 22 days after challenge. In addition, this group of animals mounted a significant immune response to chlamydial antigens, as shown by a lymphocyte proliferation assay, compared with the sham-immunized nonchallenged mice. After intrabursal challenge, there was no C. trachomatis shedding from the vagina in the C. trachomatis-immunized, C. trachomatis-challenged animals, while 63% of the sham-immunized, C. trachomatis-challenged mice had a positive C. trachomatis culture. In addition, histological sections from the genital tract showed, at 2 weeks postchallenge, a marked acute inflammatory reaction in the sham-immunized, C. trachomatis-challenged animals while in the C. trachomatis-immunized, C. trachomatis-challenged mice there was minimal inflammatory reaction. When the animals were mated, only 12% of the mice from the sham-immunized, C. trachomatis-challenged mice were fertile. In contrast, 94 and 80% of the sham-immunized, nonchallenged and C. trachomatis-immunized, C. trachomatis-challenged mice, respectively, were fertile.

Administration, Intranasal↗
PubMed1994

Induction of infertility by the Chlamydia trachomatis mouse pneumonitis biovar in strains of mice that differ in their response to the 60 kDa heat shock protein.

To determine the role that the host response to the chlamydial 60 kDa heat shock protein (hsp) plays in the pathogenesis of infertility, C3H/HeN (H-2k) and C57BL/6 (H-2b) mice were inoculated in the left ovarian bursa with 1 x 10(5) inclusion forming units of the Chlamydia trachomatis mouse pneumonitis (MoPn) biovar, and in the right ovarian bursa with mock-infected HeLa-229 cell extracts. Control mice were inoculated with mock-infected HeLa-229 cell extracts. These two strains of mice were chosen because the C3H mice mount a strong immune response to the 60 kDa hsp, whereas the C57BL/6 mice respond only weakly. Vaginal cultures obtained after inoculation were positive for 4 weeks in both strains of mice. Histological sections showed a marked acute inflammatory infiltrate that permeated all the layers of the oviduct and lasted for approximately 2 weeks in both strains. By the third week, mononuclear inflammatory cells were also observed and from 4 weeks after inoculation, hydrosalpinx formation was observed, particularly in the C3H mice. An inclusion immunofluorescence assay detected antibodies specific for chlamydia in the serum and the vaginal washes of the C3H and C57BL/6 mice. Western blot analysis of the serum samples showed an immune response to lipopolysaccharide, and the 30, 40 (major outer membrane protein) and 60 kDa cysteine-rich protein in both strains of mice. In addition, in the C3H mice a strong immune reaction was mounted against a 50 kDa component and the 60 kDa hsp. Six weeks after inoculation, the female mice were mated with male mice of proven fertility and the outcome of the pregnancies evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Phylogenetic analysis of the outer-membrane-protein genes of Chlamydiae, and its implication for vaccine development.

Examination of 18 complete and 6 partial sequences of the major outer-membrane protein from 24 chlamydiae isolates was used to reconstruct their evolutionary relationships. From this analysis, assuming that the clades with 100% bootstrap support are correct, come the following conclusions: (1) The tree of these sequences is not congruent with the phylogeny of the hosts, and thus host switching would seem to have occurred, thereby limiting the extent to which there has been coevolution of parasite and host. (2) The tree is also noncongruent with clustering by type of cell infected, thereby limiting the extent to which there has been coevolution of parasite and the cell type that it infects. (3) The tree is also noncongruent with clustering by the organ infected (eyes or genitalia), thereby limiting the extent to which there has been coevolution of parasite and the organ that it infects. (4) The tree is also noncongruent with genital strains arising from lymphogranuloma venereum strains. (5) The tree is also noncongruent with the geographic site at which the isolates were obtained, thereby limiting the extent of divergence explained by geographic separation. (6) There are estimated to be 185 amino acid positions that are invariable (as opposed to unvaried) in the major outer-membrane protein. There are 10 unvaried positions in the variable domains, of which 9 appear to be invariable, giving some reason to hope that development of a vaccine might be possible. (7) The rate of change of this protein is too small to see increased divergence over the time span of isolation of these genes, giving hope to any vaccine having longevity. Bootstrapping supports those portions of the tree on which the first five conclusions above depend. The picture that these results provide is more one of pathogen versatility than one of coevolutionary constraints. In addition, we examined 10 60-KDa, outer-membrane protein-2 genes, all but one of which were from these same strains. The tree was not, among the trachomatis strains, congruent with the major-outer-membrane protein tree, suggesting that gene exchange could be occurring among strains. Moreover, there is an apparent slowdown in divergence in this gene, among the trachomatis strains.

Amino Acid Sequence↗
PubMed1993

Mapping of a surface-exposed B-cell epitope to the variable sequent 3 of the major outer-membrane protein of Chlamydia trachomatis.

A B-cell epitope, AEFPLDIT, was located to the variable sequent 3 of the major outer-membrane protein (MOMP) using the monoclonal antibody L3-1, raised to the Chlamydia trachomatis serovar L3 MOMP. By Western blot and inclusion immunofluorescence assay the monoclonal antibody recognized all the C complex and C-related complex serovars of C. trachomatis, except serovar C. Dot-blot and ELISA data using native elementary bodies indicated that the epitope was surface exposed. The monoclonal antibody, at concentrations of 10 and 100 micrograms per 10(7) chlamydial inclusion-forming units, was able to neutralize the infectivity of chlamydia in an in vivo assay but did not neutralize chlamydia in vitro or in a mouse toxicity assay. A peptide corresponding to the variable sequent 3 has previously been shown to also elicit a T-cell response; thus, careful consideration should be given to inclusion of this region of the major outer-membrane protein in a subunit vaccine.

Amino Acid Sequence↗
PubMed1993

Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars.

A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56 degrees C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100% survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of L3 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.

Amino Acid Sequence↗
PubMed1993

Comparison of the Clearview Chlamydia, the PACE 2 assay, and culture for detection of Chlamydia trachomatis from cervical specimens in a low-prevalence population.

The Clearview Chlamydia assay (Wampole Laboratories, Cranbury, N.J.), the PACE 2 DNA probe assay (GenProbe, San Diego, Calif.), and culture were compared for their abilities to detect Chlamydia trachomatis from cervical specimens in a population with a low prevalence (3.9%) of chlamydial infections. A consensus reference method was used. The consensus reference method defined a positive specimen as one with a positive culture result or positive by both of the two nonculture methods. Of the 940 specimens tested, 37 were positive; 36 were positive by culture, 28 were positive by the PACE 2 assay, and 27 were positive by the Clearview assay, giving sensitivities of 97.3, 75.5, and 72.9%, respectively, and specificities of 100, 97.1, and 98.9%, respectively. There was a direct correlation between the number of inclusion-forming units detected by culture and the ability of the two nonculture methods to detect the positive specimens.

Adult↗
PubMed1992

Synthesis and biological evaluation of 5'-sulfamoylated purinyl carbocyclic nucleosides.

The first series of 5'-sulfamoylated carbocyclic purinyl nucleosides was synthesized and tested for antitumor and antibacterial activities. The target compounds were formed by reacting the 2',3'-acetonide-protected carbocyclic nucleosides with sulfamoyl chloride, followed by deprotection. The agents were tested for cytotoxic activity against P388 mouse leukemia cells. Two compounds, 5'-sulfamoyl carbocyclic adenosine (2) and 5-sulfamoyl-8-aza carbocyclic adenosine (6) exhibited IC50 values as low as 62 and 15 nM, respectively. These analogs inhibited protein biosynthesis and slowed down DNA and RNA biosyntheses in the P388 cells. None of the target molecules were as potent against Escherichia coli as they were against the tumor cells. Also, in cell-free systems, agents 2 and 6 were more effective inhibitors of protein synthesis in rabbit reticulocyte lysate than in E. coli. These new carbocyclic derivatives appear to be somewhat selective for eukaryotic over prokaryotic cells in affecting translation.

AMP Deaminase↗
PubMed1992

Characterization of the humoral response induced by a peptide corresponding to variable domain IV of the major outer membrane protein of Chlamydia trachomatis serovar E.

A 30-amino-acid peptide corresponding to variable domain IV (VD IV) of the major outer membrane protein of Chlamydia trachomatis serovar E was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize mice. The resulting antisera (anti-KLH-VD IV sera) recognized all 15 serovars of C. trachomatis when assayed by indirect immunofluorescence and Western blotting. Probing of overlapping hexameric peptides representing VD IV with mouse anti-KLH-VD IV sera revealed that two main regions of the peptide were recognized by the antisera, the N terminus of the peptide, which contains B-complex-specific epitopes, and the middle region of the peptide, which contains a species-conserved domain. When used in an in vitro neutralization assay, these antisera were able to neutralize mainly serovars in the B complex. These data provide evidence that a linear peptide corresponding to VD IV can induce in vitro protection from C. trachomatis infectivity that is subspecies specific.

Animals↗
PubMed1992

Detection of bacteriuria and pyuria by URISCREEN a rapid enzymatic screening test.

A multicenter study was performed to evaluate the ability of the URISCREEN (Analytab Products, Plainview, N.Y.), a 2-min catalase tube test, to detect bacteriuria and pyuria. This test was compared with the Chemstrip LN (BioDynamics, Division of Boehringer Mannheim Diagnostics, Indianapolis, Ind.), a 2-min enzyme dipstick test; a semiquantitative plate culture method was used as the reference test for bacteriuria, and the Gram stain or a quantitative chamber count method was used as the reference test for pyuria. Each test was evaluated for its ability to detect probable pathogens at greater than or equal to 10(2) CFU/ml and/or greater than or equal to 1 leukocyte per oil immersion field, as determined by the Gram stain method, or greater than 10 leukocytes per microliter, as determined by the quantitative count method. A total of 1,500 urine specimens were included in this evaluation. There were 298 specimens with greater than or equal 10(2) CFU/ml and 451 specimens with pyuria. Of the 298 specimens with probable pathogens isolated at various colony counts, 219 specimens had colony counts of greater than or equal to 10(5) CFU/ml, 51 specimens had between 10(4) and 10(5) CFU/ml, and 28 specimens had between 10(2) and less than 10(4) CFU/ml. Both the URISCREEN and the Chemstrip LN detected 93% (204 of 219) of the specimens with probable pathogens at greater than or equal to 10(5) CFU/ml. For the specimens with probable pathogens at greater than or equal to 10(2) CFU/ml, the sensitivities of the URISCREEN and the Chemstrip LN were 86% (256 of 298) and 81% (241 of 298), respectively. Of the 451 specimens with pyuria, the URISCREEN detected 88% (398 of 451) and Chemstrip LN detected 78% (350 if 451). There were 204 specimens with both greater than or equal to 10(2) CFU/ml and pyuria; the sensitivities of both methods were 95% (193 of 204) for these specimens. Overall, there were 545 specimens with probable pathogens at greater than or equal to 10(2) CFU/ml and/or pyuria. The URISCREEN detected 85% (461 of 545), and the Chemstrip LN detected 73% (398 of 545). A majority (76%) of the false-negative results obtained with either method were for specimens without leukocytes in the urine. There were 955 specimens with no probable pathogens or leukocytes. Of these, 28% (270 of 955) were found positive by the URISCREEN and 13% (122 of 955) were found positive by the Chemstrip LN. A majority of the false-positive results were probably due, in part, to the detection of enzymes present in both bacterial and somatic cells by each of the test systems. Overall, the URISCREEN is rapid, manual, easy-to-perform enzymatic test that yields findings similar to those yielded by the Chemstrip LN for specimens with both greater than or equal to 10(2) CFU/ml and pyuria or for specimens with greater than or equal to 10(5) CFU/ml and with or without pyuria. However, when the data were analyzed for either probable pathogens at less 10(5) CFU/ml or pyuria, the sensitivity of the URISCREEN was higher (P less than 0.05).

Bacteriological Techniques↗
PubMed1992

Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis.

The Mycotrim Triphasic flask system (Irvine Scientific, Irvine, Calif.) was compared with a system composed of Mycotrim GU broth (Irvine Scientific) and A7 or A8 agar (Remel, Lenexa, Kans.) for the ability to detect Ureaplasma urealyticum and Mycoplasma hominis from 129 genital specimens. Of the 64 specimens positive for U. urealyticum, 25, 98, and 100% were detected on Mycotrim Triphasic agar and A7 and A8 agars, respectively. All 18 specimens that grew M. hominis were detected by A7 and A8 agars, and 94% grew on Mycotrim Triphasic agar. Mycotrim GU broth detected all of the positive specimens, and Mycotrim Triphasic broth detected all but one. Mycotrim GU broth inoculated simultaneously with either A7 or A8 agar was found to be more sensitive and cost-effective than the Mycotrim Triphasic flask system.

Culture Media↗
PubMed1992

Identification of Mycobacterium tuberculosis and Mycobacterium avium-M. intracellulare directly from primary BACTEC cultures by using acridinium-ester-labeled DNA probes.

Identification of members of the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex (MAC) directly from primary BACTEC cultures was evaluated by using acridinium-ester-labeled DNA probes (AccuProbe; GenProbe, Inc., San Diego, Calif.). In preliminary experiments, blood present in samples was found to interfere with the assay because of nonspecific chemiluminescence, which was measured in relative light units (RLUs). There was a direct relationship between the age of the culture and the number of nonspecific RLUs. A protocol using 1% sodium dodecyl sulfate-5 mM EDTA to treat BACTEC broth cultures which, with specimens containing blood, gave on the average a ninefold reduction in nonspecific chemiluminescence was developed. By using this treatment protocol, 120 specimens were tested directly from BACTEC broth cultures with an AccuProbe for the M. tuberculosis complex and/or the MAC. In order to establish the background of the specimen, the patient sample was assayed without probe. The criteria for the inclusion of BACTEC cultures in the evaluation were a growth index of greater than or equal to 100 and a positive smear for acid-fast bacilli directly from the BACTEC broth. For the 120 cultures tested, if a hybridization result of greater than or equal to 30,000 RLUs was considered positive, the sensitivities for detecting the M. tuberculosis complex and the MAC were 47 and 90%, respectively, with a specificity of 100% for both. However, if a ratio of the RLUs obtained with the MAC or the M. tuberculosis complex probe to those obtained with the specimen background of >/= 20 was considered positive, this gave 77% sensitivity and 100% specificity for BACTEC cultures containing M. tuberculosis complex isolates and 96% sensitivity and 100% specificity for those growing MAC isolates.

Acridines↗
PubMed1991

Sequence of the gene encoding the major outer membrane protein of the mouse pneumonitis biovar of Chlamydia trachomatis.

The gene encoding the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar was sequenced and the amino acid sequence deduced. The primary structure of this protein is similar to that of the lymphogranuloma venereum and trachoma biovars in that it consists of four variable domains interspersed with five constant domains. This protein may be an ideal candidate for a vaccine in chlamydia-infected mouse experimental models.

Amino Acid Sequence↗