Preparation of mango peel-waste pectin-based nanofibers by solution blow spinning (SBS).

An essential prerequisite for successful solution blow spinning (SBS) is the presence of effective molecular entanglements of polymers in the solution. However, the fabrication of biopolymer fibers is not as straightforward as synthetic polymers. Particularly for biopolymers such as pectin, molecular entanglements are essential but insufficient for successful spinning through the SBS production method. Such a challenge is due to the biopolymer's complex nature. However, incorporating an easily spinnable polymer precursor, such as polyacrylonitrile (PAN), to pectin effectively enabled the production of fibers from the SBS process. In this process, PAN-assisted pectin nanofibers are produced with average diameters ranging from 410.75 ± 3.73 to 477.09 ± 6.60 nm using a feed flow rate of 5 ml h-1, air pressure of 3 bars, syringe tip to collector distance at 30 cm, and spinning time of 10 min. PAN in DMSO solvent at different volume ratios (i.e. 35%-55% v/v) was critical in assisting pectin to produce nanofibers. The addition of a high molecular weight polymer, PAN, to pectin also improved the viscoelasticity of the solution, eventually contributing to its successful SBS process. Furthermore, the composite SBS-spun fibers obtained suggest that its formation is concentration-dependent.

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA.

BACKGROUND: The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS: Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS: ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION: Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

The role of BRAF mutations in primary melanoma growth rate and survival.

BACKGROUND: The clinical behaviour and prognosis of primary melanomas harbouring BRAF mutations is not fully understood. OBJECTIVES: To investigate the effect of mutation status on primary melanoma growth rate and melanoma-specific survival (MSS). METHODS: A prospective cohort of 196 patients with stage I-III primary cutaneous melanoma were followed for a median of 92 months, pre-dating the institution of BRAF inhibitor therapy. Clinicopathological variables were correlated with mutation status and hazard ratios (HRs) estimated for MSS. RESULTS: Of 196 tumours, 77 (39.2%) were BRAF V600E, 10 (5.1%) BRAF V600K and 33 (16.8%) were NRAS mutant. BRAF V600E mutant melanomas were associated with favourable clinical characteristics and tended to be slower growing compared with BRAF V600K, NRAS mutant or BRAF/NRAS wild-type tumours (0.12 mm per month, 0.61 mm per month, 0.36 mm per month and 0.23 mm per month, respectively; P = 0.05). There were 39 melanoma deaths, and BRAF mutant melanomas were associated with poorer MSS in stage I-III disease [HR 2.60, 95% confidence interval (CI) 1.20-5.63; P = 0.02] and stage I-II disease (HR 3.39, 95% CI 1.12-10.22; P = 0.03) after adjusting for other prognostic variables. Considered separately, BRAF V600E mutant melanomas were strongly associated with MSS independently of thickness and nodal status (HR 3.89, 95% CI 1.67-9.09; P < 0.01) but BRAF V600K mutant tumours were not (HR 1.19, 95% CI 0.36-3.92; P = 0.77). CONCLUSIONS: The presence of a BRAF mutation does not necessarily 'drive' more rapid tumour growth but is associated with poorer MSS in patients with early-stage disease.

Telomere shortening and apoptosis in telomerase-inhibited human tumor cells.

Despite a strong correlation between telomerase activity and malignancy, the outcome of telomerase inhibition in human tumor cells has not been examined. Here, we have addressed the role of telomerase activity in the proliferation of human tumor and immortal cells by inhibiting TERT function. Inducible dominant-negative mutants of hTERT dramatically reduced the level of endogenous telomerase activity in tumor cell lines. Clones with short telomeres continued to divide, then exhibited an increase in abnormal mitoses followed by massive apoptosis leading to the loss of the entire population. This cell death was telomere-length dependent, as cells with long telomeres were viable but exhibited telomere shortening at a rate similar to that of mortal cells. It appears that telomerase inhibition in cells with short telomeres lead to chromosomal damage, which in turn trigger apoptotic cell death. These results provide the first direct evidence that telomerase is required for the maintenance of human tumor and immortal cell viability, and suggest that tumors with short telomeres may be effectively and rapidly killed following telomerase inhibition.

Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1.

The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.

A mammalian telomerase-associated protein.

The telomerase ribonucleoprotein catalyzes the addition of new telomeres onto chromosome ends. A gene encoding a mammalian telomerase homolog called TP1 (telomerase-associated protein 1) was identified and cloned. TP1 exhibited extensive amino acid similarity to the Tetrahymena telomerase protein p80 and was shown to interact specifically with mammalian telomerase RNA. Antiserum to TP1 immunoprecipitated telomerase activity from cell extracts, suggesting that TP1 is associated with telomerase in vivo. The identification of TP1 suggests that telomerase-associated proteins are conserved from ciliates to humans.

Human telomerase contains evolutionarily conserved catalytic and structural subunits.

We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits.

Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene.

The megakaryocyte growth and development factor (MGDF) is a cytokine that regulates megakaryocyte development and is a ligand for the MPL receptor. In this study, we describe the genomic structure of the human MGDF gene. The MGDF gene was found to consist of seven exons and six introns spanning 8 kilobases. The protein is encoded by exons 3 through 7. The human MGDF gene has been mapped to chromosome 3q26.3. In addition to the previously described full-length cDNA, two cDNA variants were isolated from human fetal liver. Comparison of these two cDNA sequences with the genomic sequence indicates that they arise by differential splicing.

Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)

Prokaryotic peptides that block leukocyte adherence to selectins.

Pertussis toxin binds target cells through the carbohydrate recognition properties of two subunits, S2 and S3, which share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family. Selectins appear on inflamed endothelial cells and promote rolling of leukocytes by reversibly binding carbohydrates. S2, S3, and synthetic peptides representing their carbohydrate recognition domains competitively inhibited adherence of neutrophils to selectin-coated surfaces and to endothelial cells in vitro. These proteins and peptides also rapidly upregulated the function of the leukocyte integrin CD11b/CD18. These findings implicate mimicry of eukaryotic selectins by prokaryotic adhesive ligands and link the mechanisms underlying leukocyte trafficking to microbial pathogenesis.

Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages.

Pertussis toxin (PT) and filamentous hemagglutinin can each mediate the association of Bordetella pertussis with human macrophages. Adherence via filamentous hemagglutinin leads to integrin-mediated entry and survival of the bacteria within the human cell. We determined the contribution of PT to bacterial adherence to human macrophages. Plating macrophages on wells coated with recombinant PT subunit 2 (S2) or S3 decreased PT-dependent bacterial binding by greater than 60%; S1, S4, and S5 were ineffective. S3-dependent adherence was reduced 63% +/- 8% by sialic acid, while S2-dependent adherence was reduced 53% +/- 11% by galactose. Loss of the carbohydrate recognition properties of S2 by deletion of residues 40 to 54 or site-specific mutations at Asn-93, His-47, or Arg-50 eliminated the ability of the subunit protein to competitively inhibit bacterial binding. Peptides corresponding to residues 28 to 45 of S2 and S3 competitively inhibited adherence. Treatment of macrophages with antibodies to Le(a) or Le(x) but not CD14, CD15, CD18, or HLA interfered with PT-mediated binding. Exposure of the macrophages to the B oligomer, S2, or S3 increased binding to the CD11b/CD18 integrin. These results indicate that the carbohydrate recognition domains of both S2 and S3 participate in adherence of B. pertussis to human macrophages. The PT receptor(s), as yet unidentified, appears to carry the Le(a) or Le(x) determinants and is functionally capable of modulating integrin-mediated binding to the macrophage.

Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli.

The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation. The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer. Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin. These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component.

Pertussis toxin has eukaryotic-like carbohydrate recognition domains.

Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.

Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity.

Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a region of nearly identical amino acid sequence near the NH2 terminus. Previous investigations have shown that substitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates its enzyme activity. We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxin subunit A leads to a similar loss of catalytic activity. This result suggests a correlation of function with structure between the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of a vaccine containing an enzymatically inert analog of cholera toxin.

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin.

An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.