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At least 37 records · Page 2Linked to original sources
PubMed1994

Nonrepresentative PCR amplification of variable gene sequences in clinical specimens containing dilute, complex mixtures of microorganisms.

PCR amplification and DNA sequencing of the expression locus from Neisseria gonorrhoeae contained in urine sediments collected from experimentally infected human subjects produced two observations. First, different pilin sequences were obtained when separate aliquots of the same sample were amplified and sequenced. In contrast, the same pilin sequence was obtained when repeated amplifications were performed on individual colonies grown from the clinical samples. Second, mixed sequences (i.e., more than one nucleotide at variable positions in the pilin gene sequence) were observed in both the direct clinical isolates and individual cultures grown from the isolates. These results suggest that when clinical samples are directly examined by PCR amplification and sequencing, multiple amplifications may be required to detect sequence variants in the sample and minority variant sequences will not always be detected.

Adolescent↗
PubMed1991

Effectiveness in inhibition of recovery of cell survival by cisplatin and carboplatin: influence of treatment sequence.

Clinical protocols have been designed to combine platinum-based drugs and radiation in the treatment of cancer. The rationale for this approach has been developed from preclinical studies demonstrating that platinum compounds can potentiate the cytotoxic effects of radiation toward cells. In the present study multicellular spheroids derived from squamous cell carcinoma cell line HN-1 have been used to study the effects of both cisplatin and carboplatin when administered prior to, concurrently, and after irradiation treatment. To study the influence of platinum compounds on sublethal damage repair, single and split doses of radiation were applied. Growth delay and proportion cured spheroids served as endpoints. Both cisplatin and carboplatin had no potentiating effect when administered 24 hr prior to irradiation. When administered 3 hr after completion of irradiation procedures, growth delay after single and split doses were enhanced to the same extent. The drug enhancement ratio for cisplatin was larger (1.5) than for carboplatin (1.2). Both single and split doses were enhanced by the same factor, which was interpreted as no effect on sublethal damage repair. When platinum compounds were present in the target cells at the time of irradiation, especially the split dose radiation response was strongly enhanced: the drug enhancement ratio was 3.9 for cisplatin and 3.2 for carboplatin. Recovery from sublethal damage was totally repressed. This study shows that platinum compounds can potentiate radiation and that for maximum effect the sequence of the two treatment modalities is of utmost importance. Moreover, these results may in part explain the heterogeneous outcomes of trials combining platinum compounds and radiation.

Carboplatin↗
PubMedDate not supplied

Correction of CSF motion artifact on MR images of the brain and spine by pulse sequence modification: clinical evaluation.

A modification of the standard spin-echo pulse sequence designed to suppress motion artifacts was clinically evaluated on T2-weighted MR images of the cervicocranial region. A retrospective study involving 40 patients, half of whom were examined with a standard T2-weighted multislice spin-echo sequence and half of whom were examined with a gradient waveform modification of the same sequence, uniformly demonstrated restoration of CSF signal intensity on images obtained with the gradient modified sequence. The cervical subarachnoid spaces, cisterna magna, medullary cistern, pontine cistern, fourth ventricle, and aqueduct were more consistently and brightly represented. However, the phase-encoding artifacts arising from CSF motion were not significantly reduced by using the gradient waveform modified pulse sequence. Digital subtraction of an image obtained with the standard sequence from an image of the same slice with the gradient modified sequence provides a direct image representation of CSF flow.

Brain↗
PubMed2001

Recurrent neural networks for predicting outcomes after liver transplantation: representing temporal sequence of clinical observations.

OBJECTIVES: This paper investigates a version of recurrent neural network with the backpropagation through time (BPTT) algorithm for predicting liver transplant graft failure based on a time series sequence of clinical observations. The objective is to improve upon the current approaches to liver transplant outcome prediction by developing a more complete model that takes into account not only the preoperative risk assessment, but also the early postoperative history. METHODS: A 6-fold cross-validation procedure was used to measure the performance of the networks. The data set was divided into a learning set and a test set by maintaining the same proportion of positive and negative cases in the original set. The effects of network complexity on overfitting were investigated by constructing two types of networks with different numbers of hidden units. For each type of network, 10 individual networks were trained on the learning set and used to form a committee. The performance of the networks was measured exhaustively with respect to both the entire training and test sets. RESULTS: The networks were capable of learning the time series problem and achieved good performances of 90% correct classification on the learning set and 78% on the test set. The prediction accuracy increases as more information becomes progressively available after the operation with the daily improvement of 10% on the learning set and 5% on the test set. CONCLUSIONS: Recurrent neural networks trained with BPTT algorithm are capable of learning to represent temporal behavior of the time series prediction task. This model is an improvement upon the current model that does not take into account postoperative temporal information.

Adult↗
PubMed1993

Simultaneous amplification of multiple HIV-1 DNA sequences from clinical specimens by using nested-primer polymerase chain reaction.

A sensitive and specific polymerase chain reaction (PCR) protocol with nested primers was developed for simultaneous amplification of three independent human immunodeficiency virus type 1 (HIV-1) DNA sequences from clinical specimens. DNA samples were first amplified with gag, pol, and env outer primer pairs and then with the corresponding three inner primer pairs in the same two-step reaction. Detection of the different amplification products was readily accomplished by simple agarose gel electrophoresis of the reaction product, even when starting with a single copy of HIV-1 DNA. Equivalent amounts of the three PCR products were generated, provided that the relative concentrations of the inner primer pairs were optimized. In addition, a beta-globin control primer pair could be conveniently included in the internal amplification step to verify that the DNA sample was suitable for PCR analysis. One nested multiplex PCR test was sufficient to detect HIV-1 DNA in all of 80 HIV-1-seropositive individuals and none of 50 HIV-1-seronegative healthy blood donors. The nested multiplex PCR procedure provides an attractive means for simple, rapid, and cost-effective direct detection of HIV-1 DNA in patient samples.

Base Sequence↗
PubMed1984

The Potter sequence: a clinical analysis of 80 cases.

Eighty cases of Potter sequence due to a renal or urologic abnormality were studied retrospectively. The abnormal renal findings were bilateral renal agenesis in 21.25%; cystic dysplasia in 47.5%; obstructive uropathy in 25%; and others in 5.25%. Fifteen patients had multiple congenital anomalies; of these three had aneuploidy, four had autosomal recessive syndromes, and eight were of unknown cause. Results of chromosome analysis in 41 patients and 21 sets of parents were abnormal in three patients, one of whom had a balanced translocation carrier parent; two additional patients and three parents had apparently balanced translocations. There was one recurrence within the study (the first child had bilateral renal agenesis and the second cystic dysplasia). The ultrasound prenatal diagnosis of the renal abnormality was made in eight cases between 18 and 34 weeks. Family histories were suggestive of an autosomal dominant gene disorder with incomplete penetrance in four of 45 families with nonsyndromic bilateral renal agenesis and cystic dysplasia. The evaluation of patients with the Potter sequence should include an examination for nonrenal defects, autopsy, chromosome analysis, and renal ultrasound or urologic evaluation of parents. Ultrasonographic prenatal monitoring of subsequent pregnancies in such families is strongly warranted because of a definite but unknown degree of recurrence risk.

Abnormalities, Multiple↗
PubMed1993

H-ras-1 gene mutations in basal cell carcinoma: automated direct sequencing of clinical specimens.

Ras oncogenes are activated by point mutations occurring in codons 12, 13 or 61 and almost any base pair mutation occurring within the first two positions of any of these codons results in activation of the gene. Although ras point mutations have been reported to occur in several skin neoplasms including squamous carcinoma, keratoacanthoma and melanoma, their frequency of occurrence in basal cell carcinoma is not known. We examined basal cell carcinomas from 13 patients for activating mutations in the H-ras-1 gene by automated direct sequencing of polymerase chain reaction assay amplified targets. We amplified sequences in exon 1 that flanked codons 12 and 13 and amplified sequences in exon 2 that flanked codon 61. The PCR products were centrifuged and directly sequenced using antisense primers in an automated sequencer using fluorescent dideoxyterminators. One tumor was found to show an activating G to A transversion in codon 13 which code for aspartic acid instead of glycine. Although H-ras mutations may be found in some skin tumors, they are not frequent in basal cell carcinomas. From this study we believe that direct sequencing of clinical material is of value and has advantages over other techniques. Additional studies need to be undertaken to understand the true clinical significance of ras mutations in basal cell carcinomas when they occur.

Base Sequence↗
PubMed1994

Optimization of PCR and automated sequencing of clinical isolates of respiratory syncytial virus.

A streamlined protocol was developed to carry out nucleotide sequence analysis on PCR products obtained from regions of the RNA genome of respiratory syncytial virus (RSV). Whole cell RNA extracted from tissue culture cells inoculated with RSV-infected nasopharyngeal aspirates was used as a template for single-tube cDNA synthesis and PCR amplification of specific regions of the RSV fusion (F) protein gene. The products were then purified using Sephacryl spin columns and digested using lambda-exonuclease to obtain single-stranded templates for analysis using dye-primer chemistry in an automated sequencer. Optimization of a range of parameters and comparison of a number of alternate procedures resulted in a final protocol that allowed the comparison of the sequences of a panel of local RSV subgroup A and B clinical isolates.

Base Sequence↗
PubMed1996

Dopamine and serotonin receptors: amino acid sequences, and clinical role in neuroleptic parkinsonism.

This review summarizes the amino acid sequences of the human dopamine and serotonin receptors and their human variants. The review also examines the receptor basis of the atypical antipsychotic drugs that elicit less parkinsonism than the typical antipsychotics. Because the dissociation constant of a drug varies with the radioligand, the dissociation constants of many neuroleptics are here summarized for the dopamine D2-, D4- and serotonin S2A-receptors using different radioligands. Radioligands of low solubility in the membrane (having low tissue/buffer partition) result in lower values for the neuroleptic dissociation constants, compared to radioligands of high membrane solubility. Such studies yield the intrinsic K value for a neuroleptic in the absence of a competing ligand. Clozapine, for example, has an intrinsic K value of 1.6 nM at the D4-receptor, in agreement with the value of 1.6 nM when directly measured with [3H]clozapine at D4. However, because clozapine competes with endogenous dopamine, the in vivo clozapine concentration to occupy 75% of the dopamine D4-receptors is derived to be approximately 13 nM. This agrees with the value of 12 to 20 nM in the plasma water (or spinal fluid) observed in treated patients. Moreover, in L-DOPA psychosis (in Parkinson's disease), the clozapine concentration for 75% blockade of D4 is predicted to be approximately 3 nM. This agrees with the value of approximately 1.2 nM observed by Meltzer et al. in plasma water (Neuropsychopharmacology, 12, 39-45 (1995)). This analysis supports the concept and practical value of the intrinsic K values. Some atypical neuroleptics (remoxipride, clozapine, perlapine, seroquel and melperone) have high intrinsic K values (ranging from 30 to 88 nM) at the D2-receptor, making them displaceable by high levels of endogenous dopamine in the caudate/putamen. In contrast, however, typical neuroleptics (i.e., those that typically cause parkinsonism) have intrinsic K values of 0.3 to 6 nM, making them less displaceable by endogenous dopamine. A relationship exists between the neuroleptic doses for rat catalepsy and the D2/D4 ratio of the intrinsic K values. Thus, the atypical neuroleptics appear to fall into two groups, those that bind loosely to D2 and those that are selective at D4.

Amino Acid Sequence↗
PubMed1993

Time-of-flight techniques. Pulse sequences and clinical protocols.

Time-of-flight, the most commonly used angiographic sequence, relies on the inflow of bright, relaxed blood into a region whose magnetization is suppressed by radiofrequency pulses. Good angiographic contrast relies on pulse rates (i. e., repetition times) that are fast enough and pulse amplitudes (i. e., flip angles) that are large enough to saturate the magnetization of the background without saturating the magnetization of entering blood.

Humans↗
PubMed2026

Library strategies differentially shape microbial, functional, and host signals in clinical metagenomic sequencing.

Metagenomic next-generation sequencing (mNGS) is increasingly used in infectious disease diagnostics, yet how library preparation shapes the microbial, functional, and host signals recovered from clinical samples remains poorly defined. Here, we performed a within-sample parallel comparison of three mNGS library preparation strategies-DNA-based libraries (DNAlib), RNA-based libraries (RNAlib), and total nucleic acid-based libraries (TNAlib)-across a diverse range of clinical specimens spanning five sample types. Using a curated clinical infectome as a benchmark, we show that library strategies are not interchangeable but capture distinct biological dimensions of the same specimen. RNAlib provided the most comprehensive standalone recovery of the clinical infectome, with improved detection of RNA viruses and cellular pathogens, enhanced resolution of resistance and virulence signals, and preservation of infection-associated host immune signatures. DNAlib showed stronger baseline recovery of DNA viruses and broader host genome coverage, whereas the TNAlib workflow evaluated here largely behaved as an intermediate strategy rather than a consistent improvement over dedicated DNA- or RNA-based workflows. Together, these results establish that the library preparation protocol is a major determinant of how clinical mNGS data should be interpreted and provide a framework for selecting sequencing strategies according to specific diagnostic and biological questions.IMPORTANCEMetagenomic sequencing is increasingly used in infectious disease research and clinical diagnostics, but different library preparation strategies may recover fundamentally different biological signals from the same sample. These signals include not only pathogens but also background microbes, microbial functional activity, and host immune-response patterns. Here, we systematically compared DNA-, RNA-, and total nucleic acid-based metagenomic sequencing libraries using the same clinical samples processed in parallel. We found that the three strategies did not provide equivalent information. RNA-based sequencing generated the most informative single-library view of infection, particularly for RNA viruses, cellular pathogens, functional microbial signals, and host immune-response patterns. DNA-based sequencing was more effective for DNA virus and host genome recovery, whereas the total nucleic acid sequencing workflow evaluated here generally behaved as an intermediate strategy. These findings show that library preparation can substantially influence the interpretation of metagenomic data.

functional characterization↗
PubMed1990

Differentiation of cytomegalovirus strains by restriction analysis of DNA sequences amplified from clinical specimens.

DNA sequences of 2.0 to 2.7 kb were amplified by polymerase chain reaction from four genes (major immediate-early, DNA polymerase, and glycoproteins B and H) of 20 distinct strains of cytomegalovirus (CMV). Analysis of the unpurified reaction products using selected restriction enzymes showed sufficient diversity of profiles among strains to differentiate all 20. Sequences suitable for restriction analysis were also amplified directly from clinical urine specimens and showed restriction profiles comparable to those of sequences amplified from corresponding CMV culture isolates. Ability to identify specific strains of CMV in amplification products facilitates molecular epidemiologic studies.

Antigens, Viral↗
PubMed1997

Antiviral susceptibilities and analysis of UL97 and DNA polymerase sequences of clinical cytomegalovirus isolates from immunocompromised patients.

Antiviral susceptibilities to ganciclovir, foscarnet, and cidofovir and sequencing of UL97 and DNA polymerase were done on 23 cytomegalovirus (CMV) isolates from 10 immunocompromised persons with end-organ CMV disease who were treated with ganciclovir alone or ganciclovir followed by foscarnet. Screening of UL97 for ganciclovir resistance mutations was done by restriction digest analysis. Of 14 isolates resistant to ganciclovir, 11 (79%) contained one or more UL97 mutations at codons known to confer resistance to this compound, and 10 (91%) had a concordant mutant pattern by restriction digest analysis. Of 9 isolates containing mutations in conserved regions of the DNA polymerase, 8 were resistant to ganciclovir, and 4 were cross-resistant to cidofovir. All isolates were susceptible to foscarnet. It is concluded that ganciclovir-resistant clinical CMV isolates may contain UL97 mutations, DNA polymerase mutations, or mutations in both genes. Ganciclovir therapy may select for CMV isolates that are cross-resistant to cidofovir.

AIDS-Related Opportunistic Infections↗
PubMed2025

Monogenic disorders associated with motor speech phenotypes in children and adolescents undergoing clinical exome sequencing.

PURPOSE: Prior studies investigating the genetic architecture of pediatric motor speech disorders (MSDs) have been limited by small sample sizes and an exclusive focus on apraxia. We aimed to identify pathogenic genomic variants associated with MSDs in a large pediatric population referred for exome sequencing (ES). METHODS: We identified pediatric patients with MSDs who had clinical ES between 2012 and 2022. The rate of pathogenic/likely pathogenic (P/LP) findings considered causative of the MSD phenotype was determined and delineated by sex and neurodevelopmental comorbidity. Gene-based burden testing compared the rate of P/LP variants in each gene in MSD cases with a comparison clinical ES cohort. RESULTS: Positive diagnostic results were detected in 527 of 2004 (26.3%) patients with MSDs, with higher diagnostic rates in females and individuals with neurodevelopmental comorbidities. P/LP sequence variants were detected in 262 genes. Gene-based case-referent burden analysis revealed that 30 genes were nominally associated with MSDs, 2 of which (SETBP1 and ADCY5) survived exome-wide correction. CONCLUSION: Over 25% of patients with MSDs were found to harbor P/LP variants in 262 genes, many of which have not previously been associated with MSDs. Potential clinical implications include early implementation of intensive speech therapy for children diagnosed with monogenic causes of MSDs.

Humans↗
PubMed2006

Polymorphism of the PE domain of PE/PE_PGRS sequences in clinical isolates of Mycobacterium bovis in Mexico.

Polymorphism of the PE domain of PE/PE_PGRS sequences was studied in Mycobacterium bovis isolates from different Mexican states. Samples were analyzed by spolygotyping and RFLP using IS6110 and a 235-bp fragment of the PE domain of PE/PE_PGRS as probes. With the PE probe, three different genotypes were observed, one being predominant in all states. These results confirm the high conservation of the PE domain and suggests a potential role for PE sequence as a stable genetic marker for bovine tuberculosis.

Animals↗
PubMed1994

Specific detection of Clostridium difficile toxin A gene sequences in clinical isolates.

The polymerase chain reaction (PCR) was used to specifically detect toxin A gene sequences of Clostridium difficile in DNA isolated from human faeces. A set of oligonucleotide primers derived from the non-repetitive region of the toxin A gene was developed to amplify a 634-bp DNA fragment. All 28 cytotoxic strains of C. difficile, previously characterized by a toxin B-PCR assay, were positive for the presence of toxin A gene sequences. No amplification products were obtained from DNAs extracted from non-toxigenic strains, strains of C. sordellii, or C. bifermentans. In addition, amplification of DNA extracted from C. difficile 8864, a strain which does not produce toxin A, resulted in multiple bands which probed negative for toxin A gene sequences. DNAs extracted from nine stool specimens which were positive for toxin B by the cytotoxicity assay and by the toxin B-PCR assay were also positive in this assay. Toxin A gene sequences were detected in DNAs obtained from 4/11 stool specimens which were negative by the toxin B cytotoxicity assay. These four specimens were from patients who had a history of relapses due to C. difficile-associated colitis, and whose stools had previously been found to be positive by the toxin B-PCR test despite no detectable toxin B in the specimens. These data indicate a comparable degree of clinical sensitivity between these two toxin-gene PCR-based assays. This rapid, sensitive and specific assay may be useful not only in the diagnosis of C. difficile infections, but also in molecular studies of the toxin A gene in C. difficile strains.

Bacterial Proteins↗
PubMed2006

Reappraisal of seventh-day syndrome following living donor liver transplantation.

Seventh-day syndrome (7DS) is characterized by sudden failure of a liver graft that had been working normally at about 1 week after transplantation, without an identifiable cause. A nonnegligible percentage of cadaveric liver transplants have shown this type of acute graft failure, whereas 7DS has not been reported after living donor liver transplantation (LDLT). Among 580 adult LDLT recipients in our institution between 1997 and 2003, 3 (0.5%) showed clinical sequences typical of 7DS. All three recipients showed similar but unique clinical sequences, consisting of initial uneventful recovery, dramatic rise of serum liver enzyme levels about 1 week later despite potent antirejection therapy, and subsequent graft loss. Liver biopsy findings were compatible with massive hemorrhagic necrosis. Sustained fever lasting for 2 days preceded deterioration of liver function. All three patients died prior to the opportunity for retransplantation. Our findings suggest that, as in cadaveric donor liver transplantation, 7DS can also occur following LDLT and that a preceding episode of sustained fever may be a prodrome of 7DS although its pathogenesis is yet poorly understood.

Adult↗
PubMed2024

Effect of Metagenomic Next-Generation Sequencing on Clinical Outcomes of Patients With Severe Community-Acquired Pneumonia in the ICU: A Multicenter, Randomized Controlled Trial.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) was previously established as a method that can increase the pathogen identification rate in patients with severe community-acquired pneumonia (SCAP). RESEARCH QUESTION: What is the impact on clinical outcomes of mNGS of BAL fluid (BALF) in patients with SCAP in the ICU? STUDY DESIGN AND METHODS: A multicenter randomized controlled open-label clinical trial was conducted in 10 ICUs. Patients were randomized in a 1:1 ratio to undergo BALF assessment with conventional microbiological tests (CMTs) only (ie, the CMT group) or BALF assessment with both mNGS and CMTs (ie, the mNGS group). The primary outcome was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a six-category ordinal scale or discharge from the ICU, whichever occurred first. RESULTS: A total of 349 patients were randomized to treatment between January 1, 2021, and November 18, 2022; 170 were assigned to the CMT group and 179 to the mNGS group. In the intention-to-treat analysis, the time to clinical improvement was better in the mNGS group than in the CMT group (10 days vs 13 days; difference, -2.0 days; 95% CI, -3.0 to 0.0 days). Similar results were obtained in the per-protocol analysis. The proportion of patients with clinical improvement within 14 days was significantly higher in the mNGS group (62.0%) than in the CMT group (46.5%). There was no significant difference in other secondary outcomes. INTERPRETATION: We found that compared with the use of CMTs alone, mNGS combined with CMTs reduced the time to clinical improvement for patients with SCAP. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR; www.chictr.org.cn/index.html; ChiCTR2000037894.

Humans↗