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PubMed2003

High versus low medium chain triglyceride content of formula for promoting short term growth of preterm infants.

BACKGROUND: In-hospital growth of most very low birth weight infants remains below the 10th percentile of reference intrauterine growth curves (Ehrenkranz 1999). To improve growth, fat is added to preterm formula in the form of medium chain triglycerides (MCT) or long chain triglycerides (LCT). MCT are easily accessible to the preterm infant with an immature digestive system while LCT are important in the development of the retina and visual acuity. Both have been incorporated into preterm formulas in varying amounts, but their effect on the preterm infant's short term growth is unclear. OBJECTIVES: To determine among preterm, formula fed infants, does high MCT as opposed to low MCT (high LCT) formula promote higher short term growth rates. SEARCH STRATEGY: MEDLINE (1966-2002), CINAHL (1982-2002), Cochrane Controlled Trials Register (The Cochrane Library, Issue 4, 2001), conference proceedings, and reference lists of articles were searched. SELECTION CRITERIA: All randomized trials comparing the effects of exclusive feeding of high versus low MCT formula (for a minimum of one week) on the short term growth of healthy, preterm infants. DATA COLLECTION AND ANALYSIS: Two reviewers assessed each study's quality and extracted data on growth parameters as well as adverse effects from included studies. All data used in analysis were continuous, and therefore weighted mean differences with 95% confidence intervals were reported. MAIN RESULTS: Eight randomized trials studying a total of 182 infants were included. There was no evidence of difference in short term growth parameters when high and low MCT formulas were compared. The meta-analysis of weight gain based on five studies yielded a WMD of -0.35 g/kg/d (95% CI -1.44, 0.74). Similarly, meta-analysis of weight gain in g/d based on two studies showed no evidence of difference (WMD 2.09 g/d, 95% CI -1.46, 5.64). Length gain, based on five studies, showed a non-significant WMD of 0.14 cm/wk (95% CI -0.04, 0.31). Head circumference gain, based on data from five studies, showed a non-significant WMD -0.03 cm/wk (95% CI -0.15, 0.08). Only one study reported skin fold thickness gain, with a mean difference -0.15 mm/wk (95% CI -0.41, 0.11), again not significant. Subgroup analyses according to % MCT in the high MCT formula, by 10% intervals, showed no evidence of effect of high MCT on short term weight gain within any subgroup. There are conflicting data (two studies) as to formula tolerance. There is no evidence of effect on incidence of necrotizing enterocolitis (NEC), based on small numbers in two trials. No studies were located addressing long term growth parameters or neurodevelopmental outcomes. REVIEWER'S CONCLUSIONS: There is no evidence of difference between MCT and LCT on short term growth, gastrointestinal intolerance, or necrotizing enterocolitis. Therefore, neither formula type could be concluded to improve short term growth or have less adverse effects. Further studies are necessary because the results from the included eight studies are imprecise due to small numbers and do not address important long term outcomes. Additional research should aim to clarify effects on formula tolerance and on long term growth and neurodevelopmental outcomes, and include larger study populations to better evaluate effect on NEC incidence.

Dietary Fats↗
PubMed2003

Early skin-to-skin contact for mothers and their healthy newborn infants.

BACKGROUND: Early skin-to-skin contact involves placing the naked baby prone on the mother's bare chest at birth or soon afterwards (< 24 hour). This could represent a 'sensitive period' for priming mothers and infants to develop a synchronous, reciprocal, interaction pattern, provided they are together and in intimate contact. Routine separation shortly after hospital birth is a uniquely Western cultural phenomenon that may be associated with harmful effects including discouragement of successful breastfeeding. OBJECTIVES: To assess the effects of early skin-to-skin contact on breastfeeding, behavior, and physiology in mothers and their healthy newborn infants. SEARCH STRATEGY: The Cochrane Pregnancy and Childbirth Group and Neonatal Group trials registers (December 2002), the Cochrane Central Register of Controlled Trials (The Cochrane Library, Issue 4, 2002), MEDLINE (1976 to 2002). SELECTION CRITERIA: Randomized and quasi-randomized clinical trials comparing early skin-to-skin contact with usual hospital care. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed trial quality and extracted data. We contacted study authors for additional information. We collected adverse effects information from the trials. MAIN RESULTS: Seventeen studies, involving 806 participants, were included. We found statistically significant and positive effects of early skin-to-skin contact on breastfeeding at one to three months postbirth (odds ratio (OR) 2.15, 95% confidence interval (CI) 1.10 to 4.22), breastfeeding duration (weighted mean difference (WMD) 41.99, 95% CI 13.97 to 70.00), maintenance of infant temperature in the neutral thermal range (OR 12.18, 95% CI 2.04 to 72.91), infant blood glucose (WMD 11.07, 95% CI 3.97 to 18.17), infant crying (OR 21.89, 95% CI 5.19 to 92.30) and summary scores of maternal affectionate love/touch (SMD 0.73, 95% CI 0.36 to 1.11) during an observed breastfeeding within the first few days postbirth. We found no statistically significant benefit of early skin-to-skin contact for other major clinical variables: breastmilk maturation, maternal chest circumference, infant heart rate. REVIEWER'S CONCLUSIONS: Limitations included the methodological quality of the studies, variations in the implementation of the intervention and outcome variability. Early skin-to-skin contact appears to have some clinical benefit especially regarding breastfeeding outcomes and infant crying and has no apparent short or long-term negative effects. Further investigation is recommended. To facilitate meta-analysis of the data, future research in this area should involve outcome measures consistent with those used in the studies included here. Published reports should also clearly indicate if the intervention was skin-to-skin contact and include means, standard deviations and exact probability values.

Breast Feeding↗
PubMed2005

Chemotherapy for advanced gastric cancer.

BACKGROUND: Gastric cancer currently ranks second in global cancer mortality. Most patients are either diagnosed at an advanced stage, or develop a relapse after apparently curative operation. Apart from supportive measures, systemic chemotherapy is the only treatment option available in this situation. OBJECTIVES: To assess the effect of chemotherapy versus best supportive care, combination versus single agent chemotherapy and different combination chemotherapy regimens in advanced gastric cancer. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (The Cochrane Library Issue 1, 2004), MEDLINE and EMBASE up to February 2004 and reference lists of articles. We also contacted pharmaceutical companies as well as national and international experts. SELECTION CRITERIA: Randomised controlled trials on systemic intravenous chemotherapy versus best supportive care, combination versus single agent chemotherapy and different combination chemotherapies in advanced gastric cancer. DATA COLLECTION AND ANALYSIS: Two authors independently extracted data. A third investigator was consulted in case of disagreements. We contacted study authors to obtain missing information. MAIN RESULTS: Chemotherapy versus best supportive care consistently demonstrated a significant benefit in terms of overall survival in favour of the group receiving chemotherapy (Hazard Ratios (HR) 0.39; 95% confidence intervals (CI) 0.28 to 0.52). Combination versus single-agent chemotherapy provides evidence for a survival benefit in favour of combination chemotherapy (HR 0.85; 95% CI 0.76 to 0.96). Numbers included in these comparisons were 184 and 1338 participants respectively. This benefit is achieved at the price of increased toxicity in the combination chemotherapy arms. When comparing 5-FU/cisplatin-containing combination therapy regimens with anthracyclines versus those without anthracyclines (HR 0.77; 95% CI 0.62 to 0.95 based on 501 participants) and 5-FU/anthracycline-containing combinations with cisplatin versus those without cisplatin (HR 0.83; 95% CI 0.76 to 0.91 based on 1147 participants), there was a significant survival benefit for regimens including 5-FU, anthracyclines and cisplatin. AUTHORS' CONCLUSIONS: Chemotherapy significantly improves survival in comparison to best supportive care. In addition, combination chemotherapy improves survival compared to single-agent 5-FU, but the effect size is much smaller. Among the combination chemotherapy regimens studied, best survival results are achieved with regimens containing 5-FU, anthracyclines and cisplatin. In this category, ECF (epirubicin, cisplatin and continuous infusion 5-FU) is tolerated best.

Anthracyclines↗
PubMed2006

[Expressed genes related to adolescent idiopathic scoliosis in spinal facet].

OBJECTIVE: To construct a subtractive cDNA library from the spinal facets of adolescent idiopathic scoliosis (AIS) patient with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to AIS in spinal facet. METHODS: mRNA was isolated separately from the convex side and concave side apical facets of an adolescent idiopathic scoliosis patient. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. (ds) cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respectively. After cDNAs were hybridized with each other twice and underwent two rounds of nested PCR, the PCR products were ligated with pDrive Cloning Vectors to set up the subtractive library. Sequence analysis was performed and the acquired data were aligned against the GeneBank nucleotide database. Furthermore, the spinal facets of 11 IS patients were collected. The techniques of immunohistochemistry and in situ hybridization were adopted in order to approve the gene expression difference. The images of immunohistochemistry and in situ hybridization were input to the image analysis system and were analyzed semi-quantitatively. RESULTS: A cDNA subtractive library of AIS in spinal facet was set up successfully with high subtractive efficiency. Beta2 Microglobulin (beta 2M) and calgranulin A (S100A8) were identified as highly differentially expressed genes in this study. CONCLUSION: All results confirm the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression can be useful for elucidating the genetic events in the development of AIS.

Adolescent↗
PubMed1992

Discovering peptide ligands using epitope libraries.

Epitope libraries are large collections of peptides. Each peptide is displayed on the surface of a bacteriophage particle and is encoded by a randomly mutated region of the phage genome, thus associating each unique peptide with the DNA molecule encoding it. Antibodies and other binding proteins are used to select specifically for rare, phage-bearing peptide ligands; sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding ligates have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for non-peptide-binding ligates. The current challenge lies in developing epitope library technology so that tight-binding peptide ligands can be detected for a wider variety of ligates, including those that recognize folded proteins. Should this be accomplished, many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers and vaccines.

Amino Acid Sequence↗
PubMedDate not supplied

High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach.

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.

Journal Article↗
PubMed2004

Role of nutritional intervention in patients treated with radiotherapy for pelvic malignancy.

Up to 12000 patients with gynaecological, urological and rectal cancer undergo radical pelvic radiotherapy annually in the UK. More than 70% develop acute inflammatory changes causing gastrointestinal symptoms during treatment because healthy bowel tissue is encompassed in the radiation field. In total, 50% go on to develop chronic bowel symptoms, which affect quality of life due to permanent changes in the small and large intestine. Nutritional intervention may influence acute and chronic bowel symptoms but the validity of the advice given to patients is not clear. To assess the incidence and significance of malnutrition and to examine the efficacy of therapeutic nutritional interventions used to manage gastrointestinal side effects in patients undergoing pelvic radiotherapy and those with chronic bowel side effects after treatment, a critical review of relevant original studies on human subjects was carried out using a specific set of mesh terms in MEDLINE and EMBASE databases and the Cochrane Library in September 2003. Full texts of all relevant articles were collected and reference lists were checked. Sources of grey literature including conference abstracts and web-based information were also reviewed. A total of 36 papers published in peer-reviewed journals between 1966 and 2003 were identified. In all, 14 randomised controlled trials, 12 prospective cohorts, four retrospective, two qualitative, one validation, one pilot study and two case reports were obtained. These included 2646 patients. Eight articles including three conference abstracts and web-based information were found. None of the studies was definitive because of weakness in methodology. No studies could be combined because the interventions and the end points were different. There is no evidence base for the use of nutritional interventions to prevent or manage bowel symptoms attributable to radiotherapy. Low-fat diets, probiotic supplementation and elemental diet merit further investigation.

Diarrhea↗
PubMed2000

Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (TRFLP) analysis.

In this report, we describe an experiment conducted at Kennedy Space Center in the biomass production chamber (BPC) using soybean plants for purification and processing of human hygiene water. Specifically, we tested whether it was possible to detect changes in the root-associated bacterial assemblage of the plants and ultimately to identify the specific microorganism(s) which differed when plants were exposed to hygiene water and other hydroponic media. Plants were grown in hydroponics media corresponding to four different treatments: control (Hoagland's solution), artificial gray water (Hoagland's+surfactant), filtered gray water collected from human subjects on site, and unfiltered gray water. Differences in rhizosphere microbial populations in all experimental treatments were observed when compared to the control treatment using both community level physiological profiles (BIOLOG) and molecular fingerprinting of 16S rRNA genes by terminal restriction fragment length polymorphism analysis (TRFLP). Furthermore, screening of a clonal library of 16S rRNA genes by TRFLP yielded nearly full length SSU genes associated with the various treatments. Most 16S rRNA genes were affiliated with the Klebsiella, Pseudomonas, Variovorax, Burkholderia, Bordetella and Isosphaera groups. This molecular approach demonstrated the ability to rapidly detect and identify microorganisms unique to experimental treatments and provides a means to fingerprint microbial communities in the biosystems being developed at NASA for optimizing advanced life support operations.

Bacteria↗
PubMed2004

Songbird genomics: methods, mechanisms, opportunities, and pitfalls.

The biology of songbirds poses fundamental questions about the interplay between gene, brain, and behavior. New tools of genomic analysis will be invaluable in pursuing answers to these questions. This review begins with a summary of the broad properties of the songbird genome and how songbird brain gene expression has been measured in past studies. Four key problems in songbird biology are then considered from a genomics perspective: What role does differential gene expression play in the development, maintenance, and functional organization of the song control circuit? Does gene regulation set boundaries on the process of juvenile song learning? What is the purpose of song-induced gene activity in the adult brain? How does the genome underlie the profound sexual differentiation of the song control circuit? Finally, the range of genomic technologies currently or soon to be available to songbird researchers is briefly reviewed. These technologies include online databases of expressed genes ("expressed sequence tags" or ESTs); a complete library of the zebra finch genome maintained as a bacterial artificial chromosome (BAC) library; DNA microarrays for simultaneous measurement of many genes in a single experiment; and techniques for gene manipulation in the organism. Collectively, these questions and techniques define the field of songbird neurogenomics.

Animals↗
PubMed2004

Statistical analysis of genomic tag data.

We present a series of statistical solutions to challenges that commonly arise in the production and analysis of genomic tag libraries. Tag libraries are collections of fragments of DNA or RNA, with each unique fragment often present in millions or billions of copies. Inferences can be made from data obtained by sequencing a subset of the library. The statistical approaches outlined in this paper are divided into three parts. First, we demonstrate the application of classical capture-recapture theory to the question of library complexity, i.e. the number of unique fragments in the library. Simulation studies verify the accuracy, for sample sizes of magnitudes typical in genomic studies, of the formulas we use to make our estimates. Second, we present a straightforward statistical cost analysis of tag experiments designed to uncover either disease-causing pathogens or new genes. Third, we develop a hidden Markov model approach to karyotyping a sample using a tag library derived from the sample's genomic DNA. While the resolution of the approach depends upon the number of tags sequenced from the library, we show via simulation that copy number alterations can be reliably detected for lengths as small as 1 Mb, even when a moderate number of tags are sequenced. Simulations predict very good specificity as well. Finally, all three of our approaches are applied to data from real tag library experiments. The hidden Markov model results are in line with what was expected from simulation, and genomic alterations found by applying the method to a cancer cell line library are confirmed using PCR. The methods and data described in this paper are contained in an R package, tagAnalysis, freely available at http://meyerson.dfci.harvard.edu/~tl974/tagAnalysis.

Journal Article↗
PubMedDate not supplied

Dictionary-based medical image I/O.

A general-purpose image I/O library is presented, whose properties derive from externally defined data dictionaries. The library has primarily been developed for image I/O in a medical image processing environment. It hence addresses all issues found in this field such as a variety of pixel types, architecture independence, automatic type conversion, and an unlimited number of image parameters. The data access routines in the library automatically convert the data types when needed. By redefining the data dictionaries, the I/O library can be tuned for other application areas. Explicit support for image compression is provided. The library is written in C++, and is available for interested parties.

Data Collection↗
PubMed2003

Insulin-sensitising drugs (metformin, troglitazone, rosiglitazone, pioglitazone, D-chiro-inositol) for polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterised by anovulation, hyperandrogenaemia and insulin resistance. Hyperinsulinaemia is known to be associated with an increase in cardiovascular risk and the development of diabetes mellitus. If insulin sensitising agents such as metformin are effective in treating features of PCOS, then they could have wider health benefits than just treating the symptoms of the syndrome. OBJECTIVES: To assess the effectiveness of insulin sensitising drugs in improving clinical and biochemical features of PCOS. SEARCH STRATEGY: We searched the Cochrane Menstrual Disorders & Subfertility Group trials register (December 2002), the Cochrane Central Register of Controlled Trials (Cochrane Library, Issue 4, 2002), MEDLINE (January 1966 to December 2002), and EMBASE (January 1985 to December 2002). SELECTION CRITERIA: Randomised controlled trials which investigated the effect of insulin sensitising drugs compared with either placebo or no treatment, or compared with an ovulation induction agent. DATA COLLECTION AND ANALYSIS: Performed by two reviewers, one blinded to information that could have identified the authors, publisher or results of each study. Fifteen trials were included for analysis, 13 of them using metformin and involving 543 participants. MAIN RESULTS: Meta-analysis showed that metformin is effective in achieving ovulation in women with PCOS with odds ratios of 3.88 (CI 2.25 to 6.69) for metformin versus placebo and 4.41 (CI 2.37 to 8.22) for metformin and clomifene versus clomifene alone. An analysis of pregnancy rates suggests a significant treatment effect for metformin and clomifene (OR 4.40, CI 1.96 to 9.85). Metformin has a significant effect in reducing fasting insulin levels (WMD -5.37, CI -8.11 to -2.63), blood pressure and low-density lipoprotein cholesterol (LDL). There was no evidence of effect on body mass index or waist:hip ratio. Metformin was associated with a significantly higher incidence of nausea, vomiting and other gastrointestinal disturbance, but no serious adverse effects were reported. REVIEWER'S CONCLUSIONS: Metformin is an effective treatment for anovulation in women with PCOS. Its choice as a first line agent seems justified, and there is some evidence of benefit on parameters of the metabolic syndrome. Ovulation rates are higher when combined with clomifene (76% versus 46% when used alone), but there is no evidence to indicate whether there is an increased multiple pregnancy rate with this combination. There is no data regarding its safety in long-term use in young women. It should be used as an adjuvant to general lifestyle improvements, and not as a replacement for increased exercise and improved diet.

Anovulation↗
PubMed1994

The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6.

A major challenge in basic and applied biological research is the engineering of small proteins with pre-determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin-6. In previous work, the 'minibody', a 61 residue polypeptide consisting of a beta-pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin-6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokine's biological activity. The approach described here could in principle be applied to other molecular targets.

Amino Acid Sequence↗
PubMed2004

Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries.

The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries. Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected. The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles. The majority of clones obtained in this study belonged to the Clostridium coccoides (C. coccoides) group, the Bacteroides group or the Lactobacillus group. Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C. coccoides and Bacteroides groups. The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries. T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data. T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.

Age Factors↗
PubMed2006

A new strategy for preparing molecular imaging and therapy agents using fluorine-rich (fluorous) soluble supports.

A convenient new strategy for producing radiolabeled compounds in high effective specific activity was developed using soluble fluorous supports. The reported methodology involves a fluorous linker group that is released from the substrate of interest upon reaction with radioiodine. The desired product can then be selectively separated from unreacted starting material and reaction byproducts using a simple fluorous solid-phase extraction procedure. The utility of this approach was demonstrated by labeling a series of benzoic acid derivatives which are commonly used to prepare molecular imaging agents. All compounds were produced in high radiochemical yields, purities, and effective specific activities. The strategy was further elaborated in that it was used to prepare a small collection of radiolabeled benzamides as a way of demonstrating the potential utility of this method for creating libraries of molecular imaging agents.

Benzamides↗
PubMed2004

Mining SAGE data allows large-scale, sensitive screening of antisense transcript expression.

As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3'UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense-antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples.

Computational Biology↗
PubMed2005

Protective human immunity as a vaccine discovery tool for falciparum malaria.

BACKGROUND: Plasmodium falciparum malaria remains a leading cause of morbidity and mortality in developing countries, and malaria-associated severe anemia is the major factor driving the high transfusion requirements in pediatric populations living in endemic areas. STUDY DESIGN AND METHODS: In this report, we identify and evaluate the targets of naturally acquired protective antibody responses in a cohort of n = 143 male volunteers residing in a P. falciparum holoendemic area of western Kenya. Volunteers were drug-cured of current malaria infection, blood was collected 2 weeks after treatment, and blood smears were collected weekly for 18 weeks. We identified and pooled plasma from the 10 most resistant (RP) and the 7 most susceptible individuals (SP) and utilized these pools in a differential screen of a P. falciparum cDNA expression library. We screened 550,000 clones and identified 7 clones that were uniquely recognized by RP but not by SP. Two clones encoded a C-terminal region polypeptide from rhoptry-associated membrane antigen (RAMA-pr), a recently described rhoptry-associated membrane antigen. RESULTS: We measured RAMA-pr antibody levels in plasma obtained 2 weeks after treatment. Individuals with detectable immunoglobulin G(1) anti-RAMA-pr (n = 24) had fewer positive blood films (p < 0.003) and 43 percent lower density of parasitemia (p < 0.02) than individuals with undetectable (n = 115) antibody levels. CONCLUSION: RAMA-pr is a rationally identified vaccine candidate preferentially recognized by antibodies produced by humans with a high level of naturally acquired resistance to P. falciparum infection. Our results demonstrate that naturally acquired protective antibody responses are useful tools to identify vaccine candidates for falciparum malaria.

Adolescent↗
PubMed2002

Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.

Chromosome Mapping↗