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PubMed2026

Development of a new recombineering system for Edwardsiella species.

Edwardsiella species are important aquaculture pathogens that also cause opportunistic infections in humans, necessitating efficient genome editing tools to study their pathogenesis and develop control strategies. In this study, we identified and characterized six endogenous recombinases pairs from Edwardsiella and its phages. Among these, the BAS_MS17 system exhibited the highest recombination efficiency in E. piscicida EIB202Δp. Extending homology arms from 150 bp to 200 bp improved editing efficiency by 2-fold, while the addition of Redg or Plug further enhanced recombination by 3-fold and 2.5-fold, respectively, without compromising accuracy (100%). More importantly, when applied to E. piscicida sdu12S, Redg or Plug improved the editing efficiency by 8-fold and 7-fold, respectively. Deletion of the phage-derived single-strand binding protein (SSB) reduced efficiency to 25% of the BAS_MS17 level, whereas expression of the endogenous RecA-family SSB (rSSB) increased recombinant yield by 5-fold, highlighting functional conservation. Furthermore, SSB proteins from heterologous hosts failed to enhance recombination efficiency. Using the optimized system, we successfully knocked out ten distinct genes, including virulence-associated loci, with editing accuracy exceeding 85%. Phenotypic analysis revealed that luxR, but not the other tested genes, contributes to biofilm formation. Virulence evaluation results showed that aroA, fur, and hfq are critical virulence-associated factors. Collectively, this streamlined recombineering system provides a simple, rapid, and efficient genetic tool for Edwardsiella, supporting mechanistic studies of virulence and the development of live attenuated vaccine candidates.

Edwardsiella piscicida↗
PubMed1995

Founder-effect speciation theory: failure of experimental corroboration.

The theory of founder-effect speciation proposes that colonization by very few individuals of an empty habitat favors rapid genetic changes and the evolution of a new species. We report here the results obtained in a 10-year-long and large-scale experiment with Drosophila pseudoobscura designed to test the theory. In our experimental protocol, populations are established with variable numbers of very few individuals and allowed to expand greatly for several generations until conditions of severe competition for resources are reached and the population crashes. A few random survivors are then taken to start a new population expansion and thus initiate a new cycle of founding events, population flushes, and crashes. Our results provide no support for the theories proposing that new species are very likely to appear as by-products of founder events.

Animals↗
PubMed1997

A novel method to assay herpes simplex virus neutralizing antibodies using BHKICP6LacZ-5 (ELVIS) cells.

A novel method for determining neutralizing serum antibody titers to herpes simplex virus type 2 (HSV-2) was developed based on reduction of infectivity in BHKICP6LacZ-5 (ELVIS) cells; baby hamster kidney (BHK) cells that have been genetically engineered to contain the Escherichia coli LacZ gene under the control of an inducible herpes simplex virus type 1 (HSV-1) promoter. The test has a semiautomated, colorimetric readout resulting in rapid, objective readings of infectivity reduction. Extent of neutralization is calculated against a calibration curve of virus infectivity generated in each run. HSV-2 neutralizing activity can be detected with serum dilutions in excess of 1:5120.

Animals↗
PubMed2005

Genetic services for familial cancer patients: a follow-up survey of National Cancer Institute Cancer Centers.

PURPOSE: Anecdotal reports suggest that the volume of services offered to individuals concerned with hereditary cancer risk has increased substantially in recent years. As a follow-up to our 1993 survey, we sought to determine how the scope and volume of genetic services has changed between 1993 and 2002. METHODS: We surveyed the 61 National Cancer Institute-designated cancer centers in operation in 2002 using an updated version of the questionnaire from 1993. Analysis included frequencies and summary statistics. RESULTS: The majority of cancer centers responding (46 of 56 centers; 82.1%) provided some genetic services for evaluation of familial cancer, which is a higher proportion than in 1993 (50%; P < .01). Almost all centers (42 of 46 centers; 91.3%) provided services not only to cancer patients and their families, but also to individuals concerned with risk, which is a change (P = .01) from 1993, when 64.7% of centers offered such services. In addition, increases have been found for most other measures of services rendered for familial genetic services. CONCLUSION: As public awareness of cancer susceptibility genes has grown markedly in recent years, the demand has also grown for genetic services to assess familial cancer risk. Major deleterious genetic mutations are rare, and much of the current research in genetic variation focuses on higher prevalence variants that carry lower risks. This may suggest that testing for mutations will move from genetics clinics to primary care and specialty practices. Thus, it is unclear whether the scope and volume of cancer center genetics services will continue to grow as rapidly as they have over the last decade.

Cancer Care Facilities↗
PubMed2003

Cystic fibrosis with normal sweat chloride concentration--case report.

Cystic fibrosis is a genetic disease usually diagnosed by abnormal sweat testing. We report a case of an 18-year-old female with bronchiectasis, chronic P. aeruginosa infection, and normal sweat chloride concentrations who experienced rapid decrease of lung function and clinical deterioration despite treatment. Given the high suspicion of cystic fibrosis, broad genotyping testing was performed, showing a compound heterozygous with deltaF508 and 3849+10 kb C-->T mutations, therefore confirming cystic fibrosis diagnosis. Although the sweat chloride test remains the gold standard for the diagnosis of cystic fibrosis, alternative diagnostic tests such as genotyping and electrophysiologic measurements must be performed if there is suspicion of cystic fibrosis, despite normal or borderline sweat chloride levels.

Adolescent↗
PubMed1975

Weight gain and heterosis at different stages of development of the rat.

Diallel crosses among four highly inbred lines of rats were made in order to evaluate their combining ability at 10 day intervals during postnatal growth. Analyses were undertaken to consider the relative magnitudes of different components of variation, including a heterotic effect, over a long developmental period. A general analysis of variance for least square means of body weights indicated that postnatal growth was influenced greatly by the maternal lines and to a lesser extent by the paternal line. Heterosis was measured as a statistically significant difference in body weight gain at 10 day intervals between the hybrid and either the averaged mean of the parent lines or the mean of the largest parent line. Of the effects analyzed, heterosis accounted for a major part of body weight variability in both sexes between 30 and 100 days of age but reached much higher levels in male than in female progeny. The heterotic response was most pronounced during the rapid growth phase; that is, between the ages of 20 to 50 days. Although interesting specific exceptions were observed, heterotic responses generally became more pronounced with increasing age through 100 days. Evidence for differential heterosis associated with particular lines was present in male progeny at growth stages immediately preceding and following the phase of most rapid growth. Specific combining ability, however, was generally lacking. Analyses of specific matings indicated that heterosis, in the lines tested, is a variable phenomenon which may be dependent upon the degree of genetic divergence between inbred parent lines and the combining ability of the genotype involved.

Age Factors↗
PubMed1994

[Recognition and identification of unknown infectious agents].

The recognition and the identification of previously unrecognized infectious agents require a multidisciplinary approach to specify the nosologic entity of the disease and the epidemiological data, especially the modes of transmission and the risk factors, as well as to discover the microorganism in the laboratory. In the past 20 years, significant breakthroughs have been achieved in cellular cultures (growth factors), in immunology (monoclonal antibodies), and moreover in molecular biology, which have been widely used in the field of infectious diseases. Whereas the classical methods used to grow microorganisms remain of major interest in many cases, innovating strategies have been recently designed to identify previously unknown pathogens. The genomic amplification by polymerase chain reaction (PCR) of highly conserved bacterial genes (as those coding for ribosomal RNA), from tissue biopsies for example, allow to recognize unknown bacteria. The evolutionary distance between a newly recognized pathogen and known microorganisms can be calculated through sequencing of these genes, as described for Rochalimaea henselae or Tropheryma whipplelii. The constitution of cDNA banks from infected tissues is also a novel approach allowing to clone and sequence viral genes, such as those from hepatitis C or from hepatitis E. In the near future, noteworthy improvements will be achieved to rapidly detect microorganisms with highly sensitive and specific tests using monoclonal antibodies, molecular probes (including branched DNA) and with PCR (including Q beta replicase and ligase chain reaction), and to determine the genetic diversity of microbial pathogens by new methods as pulse field gel electrophoresis or arbitrarily primed PCR. This will result in a better knowledge of the pathophysiology of infectious diseases, in a better recognition of a typical, previously unrecognized clinical expression of pathogenicity, and also in a more precise assessment of the actual impact of a given pathogen in human populations by highly sophisticated diagnosis tests.

Animals↗
PubMed2004

Non-invasive screening of fragile X syndrome A using urine and hair roots.

The diagnosis of fragile X A syndrome (FRAXA) during childhood depends largely on DNA-based diagnostic tests due to the lack of the specific clinical features. To determine a non-invasive screening method for fragile X syndrome, we studied the method of DNA-based diagnosis using urine or hair roots instead of routinely used peripheral blood cells. The amplification of repeat-containing alleles of FMR-1 by PCR using Pfu polymerase was applied on DNA extracted from urine sediments or hair roots of 50 and 28 normal individuals, respectively. Consistent amplification of repeat-containing DNA fragments of normal size to ethidium-visible quantities were obtained in 92% (46/50) of urine samples and 100% (28/28) of hair roots. No bands of normal size or abnormal or artificial smears were detected in two male FRAXA patients. No female samples were examined in the present study because the separation of two alleles was unsatisfactory on agarose gels with DNA from blood samples. Our results indicate that the use of hair roots in a DNA-based test constitutes a rapid, simple and less-invasive screen to diagnose males with FRAXA.

Adolescent↗
PubMed1980

Rapid development of acoustic trauma-induced audiogenic seizure risk in 3 strains of seizure-resistant mice.

Audiogenic seizure risk can be induced in genetically seizure resistant mice by exposure to an intense noise a few days prior to testing for seizure. This experiment demonstrates that the priming induced seizure risk can develop within 6-16 h after priming. It was argued that this finding suggested an alternative hypothesis of priming involving peripheral auditory mechanisms.

Acoustic Stimulation↗
PubMed2004

Rapid detection of mutations in Wilson disease gene ATP7B by DNA strip technology.

Wilson disease leads to severe hepatic and neurological pathology resulting from cellular copper overload in the respective tissue. Although the affected gene, ATP7B, has been identified, genetic testing is challenging, time-consuming and expensive. Here we describe the development and use of a novel diagnostic test for four frequent mutations (M769V, W779X, H1069Q and P1134P-fs) found in Germany and many other countries in Europe. The test is based on multiplex polymerase chain reaction and DNA strip technology and was found to be highly sensitive and specific, as well as timely and cost-effective. We conclude that this test is a useful and reliable tool to screen Wilson disease patients and their family members for these mutations and may facilitate diagnosis in this complex disease.

Adenosine Triphosphatases↗
PubMed1993

Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromes P-450 and microsomal epoxide hydrolase.

The MCL-5 cell line is a human lymphoblastoid TK+/- cell line that constitutively expresses a relatively high level of native CYP1A1, four other human cytochromes (CYP1A2, CYP2A6, CYP3A4 and CYP2E1) and microsomal epoxide hydrolase, carried as cDNAs in plasmids. The aim of this study was to evaluate this cell line for its suitability for detecting chromosomal anomalies, employing micronucleus formation in cells blocked at cytokinesis as the indicator of clastogenicity. Results from two laboratories ('ICR' and 'Swansea') using different protocols are reported. In the ICR protocol, aflatoxin B1, sterigmatocystin, benzo[a]pyrene, dibenz[a,h]anthracene, 3-methylcholanthrene, cyclophosphamide, N-nitrosodimethylamine, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline, benzidine, 2-aminofluorene, benzene, tamoxifen and omeprazole were tested and gave positive results. Anthracene, phenanthrene and pyrene were negative. In the Swansea protocol, AHH-1 cells, the parent line which constitutively expresses CYP1A1, but does not contain the genetically engineered human cytochromes or epoxide hydrolase, were tested in parallel with MCL-5 cells. Aflatoxin B1, sterigmatocystin, benzo[a]pyrene, N-nitrosodiethylamine, 2-acetylaminofluorene, benzene, omeprazole and tamoxifen were tested and gave positive results. Of these, only benzo[a]pyrene was equally potent in both cell lines. Assays of tamoxifen and omeprazole included kinetochore staining. Omeprazole, but not tamoxifen, induced a significant level of kinetochore-positive micronuclei. The detection of micronucleus formation in these genetically engineered cells appears to be a rapid, eclectic and sensitive method for screening for genotoxic activity in vitro.

Cell Division↗
PubMed2005

Genetic linkage map of medaka with polymerase chain reaction length polymorphisms.

With recent improvements in genetic and genomic infrastructures, great interest has been taken in genetic dissection of multi-factorial traits. A genetic map consisting of markers that are highly polymorphic and rapidly genotyped is essential for the genetic mapping of such a complex trait. Medaka, Oryzias latipes, is an excellent model system for genetic studies. To promote genetic mapping of complex traits in medaka we developed the first high-throughput and genome-wide marker set in the organism by using its genomic information and the bioinformatic techniques. We tested 545 primer pairs and obtained 265 co-dominant markers between two inbred strains, HNI and Hd-rR. Our map, consisting of 231 uniquely mapped markers, covers 1257.3 centimorgan (cM) of the medaka genome with an average interval distance of 5.4 cM. Furthermore, the newly designed markers were examined for polymorphisms among six medaka inbred strains: HNI, Hd-rR and four additional strains. Most of our markers are simple sequence length polymorphisms (SSLPs) and can be rapidly genotyped by an automated system under a single polymerase chain reaction (PCR) condition. Together with the genotyping data of six medaka inbred strains, our new marker set provides a powerful tool for genome-wide analysis of complex biological phenomena found widely in medaka populations.

Animals↗
PubMed2003

[Clinical and molecular diagnosis of inherited breast-ovarian cancer].

OBJECTIVE: The aim of this work was to pinpoint familial breast and/or ovarian cancer risk. Clinical cancer genetics include: diagnostic cancer genetics, cancer genetic counseling and management of women at high risk of developing breast and/or ovarian cancer. MATERIAL AND METHODS: An update of documented data was performed in order to assess our current knowledge about inherited breast-ovarian cancer. RESULTS: Most breast cancers (BC) are sporadic while 5-10% are estimated to be due to an inherited predisposition. Rare autosomal dominant alteration in two genes, BRCA1 and BRCA2, which confer a high risk of developing BC and ovarian cancer (OC), are likely to account for most families with multiple cases of early-onset BC and OC, but only 3-4% of all BC. The estimations of their prevalence and penetrance vary greatly, depending on the population studied, the study design, statistical methods and the sensitivity of technical methods for detection of mutations. Penetrance can be very high, but incomplete (maximum 80%). Penetrance and age at onset of the same mutation show great variability within and between BC families. This could be due to the effects of other genetic as well as non-genetic factors that are being studied. The heterogeneity of families makes the identification of other high-risk genes difficult. Additive effects of several susceptibility genes could explain much more BC in the general population (polygenic models). Molecular diagnoses have become feasible. Specific consultations in oncogenetics help clinicians and patients understand hereditary components, establish molecular diagnoses, provide guidelines for a better surveillance of people at high risk of developing BC, OC or both and reassure the mutation non-carriers. The strategies of management and treatment of BC and OC in mutation carriers remain under discussion and it is is difficult to choose preventive options. CONCLUSION: Inherited BC or OC due to a mutation in BRCA1 or BRCA2 are rare in the general population. However, women and men have the right to be informed of the possibilities concerning presymptomatic testing, risk assessment, surveillance, prevention and psychological support with respect to the potential benefits and limitations. Other large prospective studies are needed to validate rapidly the methods of surveillance and prevention in this group at "high risk" in order to adapt them to very large populations. However, predictive medicine should be used with care.

Breast Neoplasms↗
PubMed1977

In vitro correlates of rejection. II. Rat mixed lymphocyte reactivity in vitro and cardiac allograft acute rejection, hyperacute or accelerated rejection, and prolongation by active immunization.

The relationship of F1 hybrid to parental strain cardiac allograft rejection rates to mixed lymphocyte reactivity in vitro has been studied in 10 strain combinations crossing the major histocompatibility barrier in three different models of acute rejection, accelerated or hyperacute rejection after skin graft immunization, and attempted active enhancement using 10(7) donor strain bone marrow cells. Although high and low reactivity could be discerned between the F1 hybrid and reciprocal parental strain in three of five instances in the one-way lymphocyte culturr reaction, low reactivity was only associated with prolonged graft survival in one combination. Two strain combinations giving high in vitro lymphocyte responses were associated with easily enhanced grafts. The BN strains was a low responder in vitro in the three combinations tested and as a recipient strain, allografts could not be actively enhanced. After skin graft presensitization, BN recipients rejected grafts hyperacutely in two of three combinations and with a median survival time of 2 days in the third combination. The association of poor in vitro proliferative responses, the inability to induce enhancement, and rapid graft rejection after skin graft presensitization could be related to genetic mechanisms controlling the amount and class or subclass of antibody or to the generation of suppressor cells and remains to be determined.

Animals↗
PubMed2000

Rapid determination of zygosity and common aneuploidies from amniotic fluid cells using quantitative fluorescent polymerase chain reaction following genetic amniocentesis in multiple pregnancies.

Following second-trimester twin amniocentesis, we used quantitative fluorescent polymerase chain reaction (QF-PCR) assays and polymorphic small tandem repeats (STR) for rapid determination of zygosity and common aneuploidies from amniotic fluid (AF) cells in four pregnancies with like-sex twins, fused placentae and inconclusive chorionicity. The first and the second cases were suspected to have inadvertent sampling of the same amniotic cavity twice. The first case showed a dizygotic (DZ) pattern and repeat amniocentesis was thus avoided. The second case was monozygotic (MZ) and was complicated by discordant fetal growth and twin-twin transfusion syndrome. The third case was associated with a co-twin malformation, occipital encephalocele. DNA studies revealed MZ twinning with a discordant structural defect. The fourth case was associated with co-twin abnormalities of cystic hygroma and hydrops fetalis. DNA studies showed DZ twinning with discordant structural and chromosomal defects. The QF-PCR assay with STR has the advantages of rapid determination of zygosity and common aneuploidies in AF cells. This simple test appears to be useful in the instances of possible inadvertent puncture of the same amniotic cavity twice during amniocentesis and of discordant fetal structural and/or chromosomal abnormalities following genetic amniocentesis in multiple pregnancies with uncertain chorionicity.

Adult↗
PubMed2006

A review of the genomics of gastric cancer.

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death in the world. Over the past 2 decades, many exciting discoveries regarding the genomics of gastric cancer have been made. There are 2 distinct histologic types of gastric adenocarcinoma, and these types differ in their epidemiology, pathogenesis, genetic profile, and clinical outcome. The development of new approaches to functional genomics has significantly improved our ability to explore molecular alterations underlying gastric carcinogenesis and progression. The pathogenesis of intestinal-type gastric cancer follows a multistep progression that usually is initiated by H pylori infection. A wide range of genetic and epigenetic abnormalities including point mutation, loss of heterozygosity, microsatellite instability, and hypermethylation are described in intestinal-type gastric cancer and its precursor lesions. In contrast to the intestinal-type, diffuse-type gastric cancer is defined by a lack of precursor lesions; mutation or epigenetic silencing of the E-cadherin gene appears to be the key carcinogenic event. An improved understanding of the genomics of gastric cancer should lead to the rapid development of novel diagnostic techniques and molecular-based treatment strategies.

Adenocarcinoma↗
PubMed2003

Pharmacodynamics of linezolid in a clinical isolate of Streptococcus pneumoniae genetically modified to express lux genes.

A bioluminescent clinical isolate of Streptococcus pneumoniae was used to test the real-time effects of the oxazolidinone antibiotic, linezolid, on metabolism compared with effects on cell replication. Viable counts and bioluminescence measurements showed that linezolid has little bactericidal effect, which was similar at minimum (6 mg/L), intermediate (13 mg/L) and maximum (20 mg/L) serum concentrations. The post-antibiotic effect, however, was shorter when measured by light output than by viable counts. The results demonstrate that bioluminescence provides a rapid and sensitive means of measuring the effect of antimicrobials on bacterial metabolism, and that the latter recovers earlier than commencement of cell replication after linezolid exposure.

Acetamides↗
PubMed2000

Fulminant case of hereditary neuropathy with liability to pressure palsy.

Hereditary neuropathy with liability to pressure palsy (HNPP) is typified as isolated nerve palsies caused by trivial compression or trauma. It rarely presents in two extremities and even more infrequently affects all four limbs simultaneously. We present a patient who concurrently experienced right shoulder, left hand, and bilateral foot weakness mimicking several multifocal conditions. Electromyography suggested HNPP and subsequent nerve biopsy and genetic testing were confirmatory. The case demonstrates that HNPP can present in a fulminant manner and should be included in the differential diagnosis of acute multiple mononeuropathies. The possible causes for such a rapid clinical course in our patient are discussed.

Adult↗