Biomedical subjects

S E Mitchell

Publications and source records attributed to S E Mitchell.

At least 19 recordsLinked to original sources

PubMed2006

Parasite-mediated selection and the role of sex and diapause in Daphnia.

To gain insight into parasite-mediated natural selection, we studied a natural population of the crustacean Daphnia magna during a severe epidemic of the bacterial parasite Pasteuria ramosa. We also investigated the relationship between susceptibility and the production of resting eggs, which are only produced during the sexual phase of reproduction. Live host samples were taken before and after this epidemic and resistance to P. ramosa was examined in the laboratory. Host clones collected after the epidemic were more resistant to P. ramosa than were those collected pre-epidemic, which is consistent with parasite-mediated selection. In our study population, asexually reproducing females were observed across the entire study period, but females carrying resting eggs were observed only prior to the epidemic. For hosts isolated in this pre-epidemic period, we found evidence that those carrying resting eggs (at the time of collection) were more susceptible than those that were reproducing asexually. This was especially apparent for measures of parasite growth, although not all measures of infection success conclusively supported this pattern. Nevertheless, the data suggest that some genotypes invest heavily in diapause at the expense of immunocompetence. Sex could therefore inhibit the evolution of resistance because each spring new genotypes will hatch from resting eggs that are relatively susceptible as they were not exposed to the previous years bout of parasite-mediated selection.

Animals↗

PubMed2005

Diversity and selection in sorghum: simultaneous analyses using simple sequence repeats.

Although molecular markers and DNA sequence data are now available for many crop species, our ability to identify genetic variation associated with functional or adaptive diversity is still limited. In this study, our aim was to quantify and characterize diversity in a panel of cultivated and wild sorghums (Sorghum bicolor), establish genetic relationships, and, simultaneously, identify selection signals that might be associated with sorghum domestication. We assayed 98 simple sequence repeat (SSR) loci distributed throughout the genome in a panel of 104 accessions comprising 73 landraces (i.e., cultivated lines) and 31 wild sorghums. Evaluation of SSR polymorphisms indicated that landraces retained 86% of the diversity observed in the wild sorghums. The landraces and wilds were moderately differentiated (F st=0.13), but there was little evidence of population differentiation among racial groups of cultivated sorghums (F st=0.06). Neighbor-joining analysis showed that wild sorghums generally formed a distinct group, and about half the landraces tended to cluster by race. Overall, bootstrap support was low, indicating a history of gene flow among the various cultivated types or recent common ancestry. Statistical methods (Ewens-Watterson test for allele excess, lnRH, and F st) for identifying genomic regions with patterns of variation consistent with selection gave significant results for 11 loci (approx. 15% of the SSRs used in the final analysis). Interestingly, seven of these loci mapped in or near genomic regions associated with domestication-related QTLs (i.e., shattering, seed weight, and rhizomatousness). We anticipate that such population genetics-based statistical approaches will be useful for re-evaluating extant SSR data for mining interesting genomic regions from germplasm collections.

Cluster Analysis↗

PubMed2002

Evaluation of Hbr (MITE) markers for assessment of genetic relationships among maize ( Zea mays L.) inbred lines.

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker ( Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (</=0.20). Only two markers (3%) were monomorphic in all samples. Although DNA sequencing indicated that 5.5% of same-sized DNA fragments were non-homologous, this result did not affect the cluster analyses (i.e., relationships obtained from the Hbr data were congruent with those derived from pedigree information). Distance matrices generated from Hbr markers were significantly correlated ( p<0.001) with those obtained from pedigree ( r=0.782), RFLPs ( r=0.747), and SSRs ( r=0.719). Overall, these results indicated that Hbr markers could be used in conjunction with other molecular markers for genotyping and relationship studies of related maize inbred lines.

Journal Article↗

PubMed2002

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.

Arabidopsis↗

PubMed2002

Interleukin 8 in the cervix of non-pregnant ewes.

Studies of cervical artificial insemination of ewes at hormone-synchronized oestrus indicate that the cervix remains relatively impenetrable to semen, in contrast to naturally breeding animals. During parturition the inflammatory response plays an important part in cervical dilation and possibly, to a lesser extent, in the non-pregnant cervix at oestrus to facilitate the transcervical transport of semen. The expression of interleukin 8 (IL-8), a pro-inflammatory cytokine in the ovine cervix, has been mapped and quantified, using semi-quantitative in situ hybridization, to ascertain the role played by inflammation in the ovine cervix during natural and artificially induced oestrous cycles. IL-8 gene expression was observed in both the luminal epithelium and fibroblastic cells of the cervix. The presence of IL-8 was confirmed using immunohistochemistry. IL-8 gene expression in the luminal epithelium varied throughout the oestrous cycle and was highest at oestrus and at day 5 of the oestrous cycle. In ewes artificially induced to ovulate, either by the withdrawal of progesterone pessaries after treatment for 12 days, or by two i.m. injections of prostaglandin 9 days apart, IL-8 gene expression at oestrus was significantly lower than it was at natural oestrus. Insemination increased IL-8 gene expression in progesterone-synchronized ewes. These data support the hypothesis that IL-8-induced inflammation is important in normal cervical function and that this process is inhibited during artificial synchronization of the oestrous cycle and is increased by exposure to semen.

Animals↗

PubMed2001

Phase I study of twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas.

PURPOSE: To determine the maximum tolerated dose and dose-limiting toxicity associated with twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas. METHODS AND MATERIALS: Twenty-one patients with biopsy-proven adenocarcinoma of the pancreas were treated with external-beam radiotherapy to a dose of 50.4 Gy in 28 fractions, concurrent with gemcitabine, infused over 30 min before irradiation on a Monday and Thursday schedule. The dose of gemcitabine was escalated in 5 cohorts of 3--6 patients each. Initial gemcitabine dose was 10 mg/m(2), with dose escalation until dose-limiting toxicity was observed. RESULTS: The maximum tolerated dose of gemcitabine was 50 mg/m(2), when given in a twice-weekly schedule with radiation. Dose-limiting toxicity was seen in 2 patients at 60 mg/m(2), and consisted of severe upper gastrointestinal bleeding approximately 1 month after completion of treatment. Six patients had radiographic evidence of response to treatment, and 5 of these underwent complete surgical resection. Three patients who underwent complete resection had been deemed to have unresectable tumors before enrollment on trial. Four patients are alive, including 2 without evidence of disease more than 1 year after resection. CONCLUSION: The combination of external-beam radiation and twice-weekly gemcitabine at a dose of 50 mg/m(2) is well tolerated and shows promising activity for the treatment of pancreatic cancer. Our data suggest a higher maximum tolerated dose and different dose-limiting toxicity than previously reported. Further investigation of this regimen is warranted.

Adenocarcinoma↗

PubMed2001

Squamous cell carcinoma of the anal canal.

PURPOSE: To report the results of primary radiotherapy for treatment of anal canal carcinoma from the University of Florida series and review issues related to treatment of this disease. METHODS AND MATERIALS: Forty-nine patients were treated with primary radiation therapy (RT) for cure. Patients had a minimum 2-year follow-up (median, 9.8 years). After 1990, patients with lesions of at least 3 cm also received chemotherapy with fluorouracil (1000 mg/m(2)) plus cisplatin (100 mg/m(2)) or mitomycin (10-15 mg/m(2)) if medically fit (n = 26). RT was delivered with a 4-field box technique to deliver 45 Gy in 25 fractions. The inguinal nodes were treated daily using electrons to supplement the dose in that region to a total dose of 45 Gy if clinically negative or about 60 Gy if involved. There were no planned breaks. A 10- to 15-Gy boost was delivered using interstitial iridium 192 implant (n = 32), en face (60)Co field (n = 5), or external-beam photon fields (n = 11). RESULTS: Local control rates at 5 years were 100% for T1N0, 92% for T2N0 or N1, 75% for T3N0, 67% for T4N0, 88% for T4N(pos) or T(any)N2-3, and 85% overall. With surgical salvage, ultimate local control rates were 100%, 100%, 81%, 100%, and 88%, respectively, with 92% overall. Cause-specific survival rates at 5 years were 100% for Stage I, 88% for Stage II, 100% for Stage IIIA, and 70% for Stage IIIB. Absolute survival rates at 5 years were 62%, 68%, 100%, and 70%. Sphincter preservation rates were 83%, 79%, 75%, and 100% by stage and 81% overall. There was an improvement in local control with the addition of chemotherapy in more advanced disease, but it was not significant. There was an increase in acute toxicity with the addition of chemotherapy (12% > or = Grade 4) but not long-term toxicity. Late toxicity requiring colostomy occurred in 6% of patients and consisted of soft tissue necrosis. CONCLUSIONS: The majority of patients with anal canal carcinoma can be treated with curative intent using a sphincter-sparing approach of radiation with or without chemotherapy even with advanced disease. With the addition of chemotherapy to radiation, there is an increased risk of acute toxicity and about 1-2% incidence of toxic death. Smaller tumors (T1 and early T2) probably do not require the addition of chemotherapy.

Adult↗

PubMed2001

Conservative treatment of rectal adenocarcinoma.

Endocavitary radiotherapy and transrectal excision are highly effective treatments for properly selected patients with favorable early-stage rectal adenocarcinoma. The likelihood of local control and survival after treatment with either modality is similar, and differences among various series probably reflect selection. The parameter most predictive of local control and survival in the authors' series was tumor configuration. As has been previously observed, "selection is the silent partner of success." Suitable candidates for endocavitary radiotherapy or wide local excision are patients whose tumors are 3 cm or less in diameter, well-to-moderately differentiated, exophytic, mobile, limited to the submucosa on transrectal ultrasound, and within 10 cm of the anal verge. The advantages of endocavitary irradiation are (1) it is an outpatient procedure, (2) it does not require anesthesia, and (3) it is less expensive than transrectal excision. The advantages of transrectal excision are (1) it may be performed during one brief hospitalization (as opposed to four outpatient visits), and (2) a small subset of patients will have pathologic findings predicting an increased risk of regional lymph node involvement, revealing the need to treat the nodes with external-beam radiotherapy. A disadvantage of wide local excision is that some patients who would be suitable for a local procedure alone must be subjected to a course of external-beam radiotherapy when they are found to have equivocal or positive margins. Patients who are treated with transrectal excision and external-beam radiotherapy have less favorable lesions and are not comparable with patients who are treated with endocavitary radiotherapy or wide local excision alone. They are best compared with patients who have undergone major surgery consisting of abdominoperineal resection or low anterior resection. Because the risk of positive nodes is significantly increased with adverse pathologic findings such as poor differentiation, invasion of the muscularis propria, and endothelial-lined space invasion, a subset of these patients treated with wide local excision would have positive nodes. This subset of patients is not comparable with patients with stage pT1N0 and pT2N0 tumors treated with major surgery. The latter group of patients undergo complete surgical staging, whereas the pathologic staging for patients who undergo wide local excision and radiotherapy is limited to the extent of the primary tumor. With this caveat in mind, wide local excision and radiotherapy seem to result in locoregional control and survival rates similar to the rates obtained with major surgery for patients with pT1 and pT2 cancers (Table 5). Patients who should receive postoperative irradiation have tumors that exhibit one or more of the following characteristics: size greater than 3 cm in diameter, poorly differentiated, invasion of the muscularis propria, endothelial-lined space invasion, fragmented resection, equivocal or positive margins, or perineural invasion. Patients with gross residual disease are not suitable candidates for radiotherapy and require further surgery. The authors' policy is to treat these patients with chemoradiation followed by resection. Patients thought to have transmural invasion before treatment are probably best treated with preoperative chemoradiation combined with major surgery, although a subset of patients can be downstaged and rendered suitable for a wide local excision.

Combined Modality Therapy↗

PubMed2000

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis.

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.

Animals↗

PubMed2000

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Animals↗

PubMed2000