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PubMed1998

Phylogenetic relationships between prostome and colpodean ciliates tested by small subunit rRNA sequences.

We analyzed the phylogenetic relationships among the four ciliate classes, Prostomatea, Colpodea, Nassophorea, and Litostomatea, by small subunit ribosomal RNA gene sequence comparison to test several phylogenetic hypotheses that had been proposed for these classes, based on ultrastructural features. The complete coding regions of the prostomes, Prorodon teres and Coleps hirtus, and of the colpodean, Bursaria truncatella, were determined and compared to the database and the literature. The prostomes and colpodeans are strongly supported as monophyletic groups in all analyses. A sister-group relationship between prostomes and colpodeans is strongly supported by parsimony analyses and moderately supported by distance matrix analyses. There was no support for a close relationship between colpodeans and nassophoreans or between colpodeans and litostomes. In all analyses, these four classes branch late in the ciliate tree. This finding further supports the new view of ciliate phylogeny that so-called ancestral taxa, such as the "primitive" prostomes, are in fact derived and that so-called "highly evolved" groups, such as the heterotrichs, branch off very early in the ciliate tree.

Animals↗
PubMed2026

Genomic characterization of KPC-2 and NDM coproducing carbapenem-resistant Klebsiella pneumoniae in a hospital: discovery of ST1869 clone and a novel hybrid plasmid.

UNLABELLED: To characterize the plasmid architecture and molecular background of KPC-NDM coproducing carbapenem-resistant Klebsiella pneumoniae (KN-CRKP) in a South China hospital. Five KN-CRKP isolates were collected, including three from one patient. All underwent Illumina sequencing; two (ST11 and ST1869) additionally had Nanopore sequencing. Antimicrobial susceptibility testing strain sequence types, conjugation assays, resistance gene profiling, plasmid typing, genetic structure comparison, core-genome single nucleotide polymorphisms (SNPs) analysis, and plasmid clustering were performed. All isolates exhibited an imipenem minimum inhibitory concentration (MIC) of ≥128 µg/mL and harbored multiple resistance genes. One isolate (1/5) belonged to ST1869 and co-harbored blaKPC-2 and blaNDM-5. The blaNDM-5-carrying plasmid was a novel IncI1/X3 fusion plasmid that also carried blaCMY-42. Unlike several IncX3 plasmids carrying blaNDM in publicly available KN-CRKP genomes from South China, this IncI1/X3 hybrid lacked a complete conjugative transfer system. ST11 was the predominant clone (4/5), co-harboring blaKPC-2 and blaNDM-1. A rare genetic structure, ΔISKpn6-blaKPC-2-ISKpn28, was identified on IncFII plasmids carrying blaKPC-2. Plasmid clustering analysis of 126 comparative KN-CRKP genomes showed diverse sequence types and plasmid backgrounds associated with the KPC/NDM co-production pattern. The observed plasmid diversity and structural variation in KN-CRKP support continued genomic surveillance, with particular attention to the ST1869 clone, the novel IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare "ΔISKpn6-blaKPC-2-ISKpn28" genetic structure. Expanded genomic data on KN-CRKP are needed to further elucidate its resistance mechanisms and plasmid evolutionary trajectories. IMPORTANCE: The co-production of KPC and NDM carbapenemases in Klebsiella pneumoniae poses a formidable threat to clinical antimicrobial therapy, as these enzymes confer resistance to virtually all β-lactam agents, including carbapenems. Here, we report novel genomic features of KN-CRKP in South China, including the emergence of the ST1869 clone, a unique IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare ΔISKpn6-blaKPC-2-ISKpn28 genetic structure. These findings substantially expand current understanding of plasmid evolution and resistance gene dissemination in this region. The identification of diverse resistance mechanisms and clonal backgrounds supports enhanced genomic surveillance and infection-control awareness for pan-resistant Enterobacterales.

Plasmids↗
PubMed1994

Development of a nested-PCR test based on sequence analysis of epizootic hemorrhagic disease viruses non-structural protein 1 (NS1).

Two orbiviruses, epizootic hemorrhagic disease (EHD) and bluetongue (BTV) viruses, cause disease in domestic and wild ruminant species. The gene that encodes non-structural protein 1 (NS1) of EHD virus, serotype 1, was sequenced and compared to EHD and BTV NS1 sequences. The NS1 gene was found to be more conserved than the VP3 gene, and was selected as a target for polymerase chain reaction (PCR) amplification. The NS1 genes of several BTV viruses and another orbivirus, African horse sickness (AHS), were compared to the EHD NS1 genes. This information was used to develop a capture nested-PCR for detection and differentiation of EHD from BTV viral RNA.

African Horse Sickness Virus↗
PubMed1993

Priority of test locations for automated perimetry in glaucoma.

PURPOSE: Static threshold automated perimetry is a demanding test which can be tiring for some patients. The authors investigate how to optimize early stages of the test which can shorten examination time and improve performance. The effectiveness of measuring every point twice to improve diagnostic precision (proportion of eyes correctly diagnosed as normal or glaucomatous) also was evaluated. METHODS: The authors evaluated the relative contributions of individual test locations to the sensitivity and specificity of static threshold perimetry. One hundred visual fields (Octopus Program G1) of 100 patients with open-angle glaucoma and early glaucomatous defects were used to rank the most frequently defective test locations. This sequence was modified so that highly correlated points were not ranked together. The sensitivities and specificities of the defined sequence of test presentations were then measured in a separate database of 70 normal controls and 70 patients with early glaucomatous visual field defects. RESULTS: Sensitivity and specificity were, respectively, 80% and 80% after 12 locations, 89% and 89% after 26 locations, and 97% and 99% after all 59 test locations. The information obtained with the first phase alone approximates that of both phases. CONCLUSION: Staging of locations tested with automated perimetry in glaucoma may be a valuable method to reduce examination time, minimize fatigue effects, and optimize diagnostic information. Retesting every point does not improve diagnostic precision.

Glaucoma, Open-Angle↗
PubMed1987

Common and distinctive features of verbal and pictorial stimuli as determinants of psychophysiological responsivity.

The present study focuses on the relation between stimulus features and psychophysiological responsivity by using a modified version of the information detection paradigm. Compound pictorial and verbal stimuli (schematic faces with beard, glasses, and hat, and descriptions of people in terms of occupation, city of residence, a hobby, and a personality trait) were used as the relevant stimuli that subjects were instructed to memorize. Skin conductance responses were measured during the subsequent presentation of a sequence of test stimuli. Each sequence included a critical stimulus that shared from one to four common components with the relevant stimulus in each of two pictorial and two verbal experiments. We hypothesized that the electrodermal responsivity to the critical stimulus would reflect the degree it matches the relevant one. The results indicated that when the critical stimulus was identical to the relevant stimulus, responsivity was maximal. Neutral stimuli (i.e., those that shared no components with the relevant stimulus) produced minimal responsivity, and critical stimuli that only partially matched the relevant one produced intermediate levels of responsivity (in spite of the subjects' awareness of the differences between the critical and the relevant stimuli). The monotonic relation between the degree of match and responsivity supports the proposed model, which assumes that each stimulus in the sequence is being compared with the relevant stimulus by a feature-matching process, and electrodermal responsivity is related to the outcome of this process. In a fifth experiment, we compared geometric and contrast models for similarity and found that the pattern of responsivity violated the minimality and symmetry assumptions of the geometric model. The relation between cognitive processes and psychophysiological responsivity is discussed, as are implications for the application of the guilty knowledge technique for detecting information.

Arousal↗
PubMed1997

Analysis of HIV-1 env gene sequences reveals evidence for a low effective number in the viral population.

Selection is usually considered to be the dominant force controlling viral variation; the large population sizes suggest that deterministic population genetic models are appropriate. To investigate their validity for HIV, samples of env gene sequences were tested for departure from neutrality because of mutation-selection balance. None of the samples departed significantly when tested as nucleotide sequences. At the amino acid level, significantly elevated diversity was detected in two samples within, but not outside, the V3 loop. The effective population number has been estimated (using a phylogenetic method) to be close to 10(3). Estimates from nucleotide diversity are about 2-fold lower. The low value of the effective population number might arise from high variability in progeny number between infected cells, from the expansion in population number from a small inoculum as the virus is transmitted between hosts, or from variable selection at linked sites. These results suggest that the population genetics of HIV are best described by stochastic models.

Amino Acid Sequence↗
PubMed2026

A trainable language model with potential to modulate translation rates in non-model organisms by generating upstream untranslated region sequence libraries.

Tuning protein expression in non-model organisms is often constrained by the lack of validated genetic parts and predictive design tools. Translational tuning through the modulation of upstream untranslated regions (5'-UTRs) offers a potentially organism-agnostic route, but existing methods typically rely on mechanistic assumptions, prior knowledge that may not be available in non-model contexts, or the screening of sequence libraries. Here, we present a simple generative approach for creating synthetic 5'-UTR libraries based solely on the genomic sequence statistics of any desired organism. The method uses a sliding-window n-gram language model applied to native 5'-UTR sequences to produce novel sequences that preserve organism-specific base distributions and motifs without hard-coding specific motifs or mechanistic rules into inflexible statistical templates. We have applied this approach to the model bacterium Escherichia coli and the non-model probiotic Limosilactobacillus reuteri. Libraries of approximately 1,000 sequences were generated for each organism, from which about 100 unique sequences were experimentally tested for translation of a fluorescent reporter protein. In both organisms, the synthetic libraries yielded a broad range of translation levels from this relatively small number of tested variants. Sequences derived from an organism's own genomic statistics provided a more uniformly distributed range of translation rates in that organism than sequences derived from the other species. Correlations of individual sequence performance across the two species were weak, and thermodynamic predictions of ribosome binding strength showed very little predictive power, especially in the non-model L. reuteri. The results demonstrate that simple statistical language model approaches applied to genomic data can generate functional translational regulatory sequence libraries without detailed mechanistic knowledge or explicit reference to consensus motifs. The approach requires minimal computational resources, avoids reproducing native sequences, and can be readily applied to any organism with a sequenced genome. This strategy may lower technical barriers to expression tuning in non-model organisms.

5' Untranslated Regions↗
PubMed2026

Clinical implementation of next-generation sequencing in tertiary health system: the Rijeka retrospective study.

OBJECTIVE: This study aimed to assess the diagnostic yield, clinical indications, and utility of next-generation sequencing (NGS) testing since its implementation through collaboration between the University of Rijeka Faculty of Medicine and the Clinical Hospital Centre Rijeka. MATERIALS AND METHODS: This retrospective study included patients referred between 2018 and 2023 from the Clinical Hospital Centre Rijeka to the University of Rijeka Faculty of Medicine for genetic testing, primarily using exome sequencing. RESULTS: Between April 2018 and December 2023, 412 patients were referred for exome sequencing, of whom 353 (85.7%) underwent diagnostic genetic testing. A notable increase in tests ordered was observed over time. Patients were most frequently referred from Pediatrics (55.0%), Neurology (29.5%), Cardiology (7.4%), Ophthalmology (3.4%), and others (4.7%). A diagnosis was confirmed in 103/353 patients, corresponding to an overall diagnostic yield of 29.2%, and an adjusted diagnostic yield of 27.2% after collapsing related individuals into single family units. In these confirmed cases, 83 distinct disorders involving 71 unique genes were identified, with most patients showing heterozygous variants and several recurrent disorders and genes. Variants of uncertain significance were reported in 35/353 (9.9%) patients. CONCLUSION: The 27.2% diagnostic yield demonstrates effective integration of NGS into tertiary clinical practice. The recent introduction of medical genetics specialization is expected to further improve referral quality, variant interpretation, and overall diagnostic outcomes.

exome sequencing↗
PubMed1997

Polyphyly of "sclerosponges" (Porifera, Demospongiae) supported by 28S ribosomal sequences.

To test the competing hypotheses of polyphyly and monophyly of "sclerosponges," sequences from the 5' end of 28S ribosomal RNA were obtained for Astrosclera willeyana, Acanthochaetetes wellsi, and six other demosponge species. Phylogenetic relationships deduced from parsimony and neighbor-joining analyses suggest that these sclerosponges belong to two different orders of Demospongiae: Astrosclera willeyana, being closely related to the Agelasidae, belongs to the Agelasida, Acanthochaetetes wellsi, being closely related to the Spirastrellidae, belongs to the Hadromerida. These results contradict the hypothesis that sclerosponges are monophyletic and imply that a massive calcareous skeleton has evolved independently in several lineages of sponges.

Animals↗
PubMed1988

Sequencing of trimodality therapy [cis-diamminedichloroplatinum(II)/hyperthermia/radiation] as determined by tumor growth delay and tumor cell survival in the FSaIIC fibrosarcoma.

In order to improve local control of tumors over that achievable with local hyperthermia and radiation, we are testing the use of systemic cis-diamminedichloroplatinum(II) (CDDP) in conjunction with the other two modalities. In the FSaIIC fibrosarcoma, growth delay experiments indicated that the use of any two modalities resulted in at least additive effects on growth delay. When the trimodality treatment was tested, the sequence CDDP followed by hyperthermia followed by X-ray produced a growth delay of approximately 25 days which was superior to the growth delay produced by the sequences CDDP, X-ray, and hyperthermia (19 days) and X-ray, CDDP and hyperthermia (14 days). In excision experiments, also performed in the FSaIIC tumor system, we again observed clearly superior cytotoxicity in the sequence CDDP, hyperthermia, and X-rays over the other sequences tested. Our results indicate that scheduling CDDP just prior to heating and following the heat treatment with the radiation fractions results in the best tumor cell kill, probably because this sequence takes maximum advantage of the radiosensitizing properties of the combined heat-CDDP treatment. In addition, the strong cytotoxic interaction between CDDP and hyperthermia is also optimized by this scheduling. We believe these results have significant clinical implications.

Animals↗
PubMedDate not supplied

Picture and motor sequencing in Parkinson's disease.

A recent report found that the discrepancy between scaled scores on the Vocabulary and Picture Arrangement subtests of the Wechsler Adult Intelligence Scale-Revised (WAIS-R) was much larger for patients with Parkinson's disease than for healthy controls or patients with Alzheimer's disease. On this basis, it was argued that Parkinson's disease causes a specific deficit in cognitive sequencing that occurs even when the memory and speed requirements of the task are minimal. However, other work indicated that Parkinson's disease patients are almost as severely impaired on other Performance subtests from the WAIS-R that do not require sequencing. In the present study, an extremely simple, untimed test of picture sequencing and a version of the Luria three-step test of motor sequencing were employed. Parkinson's disease patients were impaired on both tasks. The extent of their impairments on picture and motor sequencing were positively correlated, and the severity of deficits on both sequencing tests was related to global mental status and to performance on the Wisconsin Card Sorting Test, but not to neurologic measures of disease severity. However, the patients' performance on the picture sequencing test but not on the motor sequencing test was related to performance on Benton's Facial Recognition Test. These results demonstrate the existence of a generalized sequencing deficit in Parkinson's disease that appears dissociable from impairment in performing simple motor acts.

Aged↗
PubMed1989

A statistical test of phylogenies estimated from sequence data.

A simple approach to testing the significance of the branching order, estimated from protein or DNA sequence data, of three taxa is proposed. The branching order is inferred by the transformed-distance method, under the assumption that one or two outgroups are available, and the branch lengths are estimated by the least-squares method. The inferred branching order is considered significant if the estimated internodal distance is significantly greater than zero. To test this, a formula for the variance of the internodal distance has been developed. The statistical test proposed has been checked by computer simulation. The same test also applies to the case of four taxa with no outgroup, if one considers an unrooted tree. Formulas for the variances of internodal distances have also been developed for the case of five taxa. Conditions are given under which it is more efficient to add the sequence of a fifth taxon than to do 25% more nucleotide sequencing in each of the original four. A method is presented for combining analyses of disparate data to get a single P value. Finally, the test, applied to the human-chimpanzee-gorilla problem, shows that the issue is not yet resolved.

Animals↗
PubMed1995

Transcription and processing of the rodent ID repeat family in germline and somatic cells.

ID elements comprise a rodent SINE (short interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected in various rat tissues, including brain and testes. We have analysed the nucleotide sequences of testes- and brain-derived ID transcripts isolated by size-fractionation, C-tailing and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA-transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable in both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification.

Animals↗
PubMed1990

Interrelationships among major protistan groups based on a parsimony network of 5S rRNA sequences.

To test the validity of the maximum parsimony approach to discern protistan interrelationships, we have derived an optimal network of 16S-like rRNA sequences using our parsimony algorithm and compared it with those reported using the distance matrix method. We have also derived an optimal network topology of 50 5S rRNA sequences through an interactive search using our algorithm. In both these networks, the kinetoplastids and euglenoids form a linkage group with Dictyostelium emerging from its neighbourhood. The cryptophytes, dinoflagellates and chromophytes and green algae emerge as independent lines suggesting that plastids arose more than once during protistan evolution. The large 5S rRNA tree further indicates independent origins of mesozoa and metazoa; kinetoplastids and ciliates; and diphyletic origin of fungi. Comparatively close positions of charales and land plants, chytrids and Zygomycetes, Physarum and amoeba, and red algae and green algae are also seen in this network.

Animals↗
PubMed1997

Differential display in primary and metastatic medullary thyroid carcinoma.

Despite recent advances in the understanding of the role of the RET proto-oncogene in the development of familial and approximately 30% of sporadic medullary thyroid carcinomas (MTC), little is known about other genetic events that modify the course and outcome of the disease. We compared the expression of genes in intrathyroidal MTCs to autologous local lymph node metastases by means of mRNA differential display (DDRT-PCR). This is the first report of differential display using surgical specimens of a primary cancer and its metastases. Total RNA was extracted from tumor tissue of two patients with MTC associated with multiple endocrine neoplasia (MEN 2B) and sporadic MTC, respectively. Following reverse transcription (RT), the products were PCR-amplified and separated on a denaturating polyacrylamide gel. RT-PCR products demonstrating differential expression were reamplified and used as probes for Northern blot analysis. Six fragments for which differential expression was confirmed were cloned and sequenced. Resultant sequences were tested for homology to sequences in public data bases, and two novel MTC-derived fragments (MDF-1, MDF-2) were identified. Sensitivity of the method was confirmed by identification of a sequence encoding the calcitonin precursor flanking peptide which is expressed almost exclusively in MTC and normal thyroid C cells. Overexpression of the ribosomal genes S3a and P0 was found in the metastases. Recent reports suggest that components of the translational apparatus act as regulatory mediators of growth, proliferation, and neoplastic change. The altered expression of ribosomal proteins and gene products encoded by MDF-1 or MDF-2 may play an important role in the progression and metastatic spread of MTC.

Blotting, Northern↗