A mutant of Haemophilus influenzae, designated HM5, carrying a mutation in the rec-1 gene region, is described. This mutant transformed approximately 100fold less well than does the wild type, but approximately 100-fold better than recl mutants. The mutant was less sensitive to UV irradiation and less "reckless" than recl mutants. In contrast to recl lysogens, HPlcl lysogens of the mutant were inducible, and during transformation, recombinant-type activity was formed to the same extent as in the wild type. Although the integration of donor DNA was complete, the integrated DNA was not replicated at 36°C. Both the inhibition of replication of the donor-recipient DNA complex and the transformation deficiency could be suppressed when, after DNA entry, the cells were incubated under suboptimal conditions. The loss of colony formation after UV irradiation was suppressible by the same conditions. 852 on July 15, 2020 by guest
A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry ofheterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.
In the HM5 mutant of Haemophilus influenzae, which carries a mutation in the rec-1 gene region and in which the replication of donor-recipient DNA complexes formed in transformation is inhibited, the transformation frequency could be greatly enhanced by inhibition of protein synthesis during transformation, indicating that transformation in the HM5 mutant induces the synthesis of a protein that inhibits the replication of the donor-recipient DNA complexes. This induction occurred in an early step of the recombination. Synthesis of the wild-type Rec-1 protein after transformation of the HM5 mutant with wild-type DNA could diminish the inhibiting effect on DNA replication. The HM5 mutant synthesized an altered Rec-1 protein (molecular weight, 38,000) whose pl differed from that of the wild type. As a result of the mutation in the rec-1 gene, two other proteins (molecular weights, 37,500 and 43,000) are lacking in the HM5 mutant.We have recently described a mutant of Haemophilus influenzae, designated HM5 (5), with properties different from two other types of mutants that carry a mutation in the rec-1 gene region: the highly recombination-deficient rec-1 mutants, transforming about 104-fold less than the wild type (9), and the almost recombinationproficient ird mutants, in which transformation is reduced only 3to 10-fold as compared with the wild type (10). The HM5 mutant, in which the mutation is also located in the rec-1 gene region, transforms approximately 100-fold less well than the wild type. The mutant is less sensitive to UV irradiation and less "reckless" than the rec-1 mutants. In contrast to rec-1 lysogens, HPlc1 lysogens of the mutant are inducible.Contrary to the rec-1 mutants and the ird mutants, the transformation deficiency in the HM5 mutant is not due to a reduced ability to form molecules with recombinant-type activity. In this mutant the integration of donor DNA in the recipient DNA is complete, but the integrated DNA is not replicated at 36°C. The inhibition of replication of the donor-recipient DNA complex and the inefficiency of transformation could be suppressed by incubation of the cells under suboptimal conditions after the entry of DNA. The loss of colony formation after UV irradiation was also suppressible by suboptimal conditions (5).This report shows that recombination in the HM5 mutant induces the synthesis of a protein which is responsible for the inhibition of DNA replication. Furthermore, evidence is presented that the mutant produces an altered Rec-1 protein. MATERIALS AND METHODSStrains. The strains used were as described previously (5, 10).Media and methods. The media and transformation assays were as described previously (6). The cells were made competent as described by Kooistra et al. (5).Protein composition in cell lysates. (i) SDS-PAA gel electrophoresis. Cells of competent cultures of an Rd strain, the HM5 mutant, and a rec-1 mutant (strain TD24 [9]) were transferred to MIc medium (2), without methionine. To 10 ml of cells 10 ,uCi of [35S]methionine (specific activity, ca. 4...
ImportanceOutcome prediction after endovascular treatment (EVT) for ischemic stroke is important to patients, family members, and physicians.ObjectiveTo develop and validate a model based on preprocedural and postprocedural characteristics to predict functional outcome for individual patients after EVT.Design, Setting, and ParticipantsA prediction model was developed using individual patient data from 7 randomized clinical trials, performed between December 2010 and December 2014. The model was developed within the Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials (HERMES) collaboration and external validation in data from the Dutch Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands (MR CLEAN) Registry of patients treated in clinical practice between March 2014 and November 2017. Participants included patients from multiple centers throughout different countries in Europe, North America, East Asia, and Oceania (derivation cohort), and multiple centers in the Netherlands (validation cohort). Included were adult patients with a history of ischemic stroke from an intracranial large vessel occlusion in the anterior circulation who underwent EVT within 12 hours of symptom onset or last seen well. Data were last analyzed in July 2022.Main Outcome(s) and Measure(s)A total of 19 variables were assessed by multivariable ordinal regression to predict functional outcome (modified Rankin Scale [mRS] score) 90 days after EVT. Variables were routinely available 1 day after EVT. Akaike information criterion (AIC) was used to optimize model fit vs model complexity. Probabilities for functional independence (mRS 0-2) and survival (mRS 0-5) were derived from the ordinal model. Model performance was expressed with discrimination (C statistic) and calibration.ResultsA total of 781 patients (median [IQR] age, 67 [57-76] years; 414 men [53%]) constituted the derivation cohort, and 3260 patients (median [IQR] age, 72 [61-80] years; 1684 men [52%]) composed the validation cohort. Nine variables were included in the model: age, baseline National Institutes of Health Stroke Scale (NIHSS) score, prestroke mRS score, history of diabetes, occlusion location, collateral score, reperfusion grade, NIHSS score at 24 hours, and symptomatic intracranial hemorrhage 24 hours after EVT. External validation in the MR CLEAN Registry showed excellent discriminative ability for functional independence (C statistic, 0.91; 95% CI, 0.90-0.92) and survival (0.89; 95% CI, 0.88-0.90). The proportion of functional independence in the MR CLEAN Registry was systematically higher than predicted by the model (41% vs 34%), whereas observed and predicted survival were similar (72% vs 75%). The model was updated and implemented for clinical use.Conclusion and relevanceThe prognostic tool MR PREDICTS@24H can be applied 1 day after EVT to accurately predict functional outcome for individual patients at 90 days and to provide reliable outcome expectations and personalize follow-up and rehabilitation plans. It will need further validation and updating for contemporary patients.
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