Method for creating polynucleotide and polypeptide sequences

The invention provides methods for evolving a polynucleotide toward acquisition of a desired property. Such methods entail incubating a population of parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes. The heteroduplexes are then exposed to a cellular DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants. The resulting polynucleotides are then screened or selected for the desired property

Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain

Background: Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP), a mild, chronic pneumonia of swine. Despite presenting with low direct mortality, EP is responsible for major economic losses in the pig industry. To identify the virulence-associated determinants of M. hyopneumoniae, we determined the whole genome sequence of M. hyopneumoniae strain 168 and its attenuated high-passage strain 168-L and carried out comparative genomic analyses. Results: We performed the first comprehensive analysis of M. hyopneumoniae strain 168 and its attenuated strain and made a preliminary survey of coding sequences (CDSs) that may be related to virulence. The 168-L genome has a highly similar gene content and order to that of 168, but is 4,483 bp smaller because there are 60 insertions and 43 deletions in 168-L. Besides these indels, 227 single nucleotide variations (SNVs) were identified. We further investigated the variants that affected CDSs, and compared them to reported virulence determinants. Notably, almost all of the reported virulence determinants are included in these variants affected CDSs. In addition to variations previously described in mycoplasma adhesins (P97, P102, P146, P159, P216, and LppT), cell envelope proteins (P95), cell surface antigens (P36), secreted proteins and chaperone protein (DnaK), mutations in genes related to metabolism and growth may also contribute to the attenuated virulence in 168-L. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for virulence. Conclusions: We studied the virulence attenuation mechanism of M. hyopneumoniae by comparative genomic analysis of virulent strain 168 and its attenuated high-passage strain 168-L. Our findings provide a preliminary survey of CDSs that may be related to virulence. While these include reported virulence-related genes, other novel virulence determinants were also detected. This new information will form the foundation of future investigations into the pathogenesis of M. hyopneumoniae and facilitate the design of new vaccines

Time to Clinical Benefit of Intensive Blood Pressure Lowering in Patients 60 Years and Older With Hypertension

Importance Recent guidelines recommend a systolic blood pressure (BP) goal of less than 150 mm Hg or even 130 mm Hg for adults aged 60 years or older. However, harms from intensive BP treatments occur immediately (eg, syncope, fall), and benefits for cardiovascular event reduction emerge over time. Therefore, harms with low chance of benefit need to be clearer, particularly for those with limited life expectancy. Objective To estimate the time needed to potentially derive clinical benefit from intensive BP treatment in patients 60 years and older. Design, Setting, and Participants This secondary analysis included individual patient data from published randomized clinical trials with 27 414 patients 60 years or older with hypertension. Patient-level survival data were reconstructed when the original data were not available. Published trials were identified by searching PubMed until October 15, 2021. Exposures Intensive BP lowering vs standard BP lowering with the treat-to-target design. Main Outcomes and Measures Major adverse cardiovascular event (MACE) defined by each trial, which was broadly similar with all trials including myocardial infarction, stroke, and cardiovascular mortality. Results Six trials (original data from 2 trials and reconstructed data from 4 trials) with 27 414 participants (mean age, 70 years; 56.3% were women) were included in the analysis. Intensive BP treatment with a systolic BP target below 140 mm Hg was significantly associated with a 21% reduction in MACE (hazard ratio, 0.79; 95% CI, 0.71-0.88; P < .001). On average, 9.1 (95% CI, 4.0-20.6) months were needed to prevent 1 MACE per 500 patients with the intensive BP treatment (absolute risk reduction [ARR], 0.002). Likewise, 19.1 (95% CI, 10.9-34.2) and 34.4 (95% CI, 22.7-59.8) months were estimated to avoid 1 MACE per 200 (ARR, 0.005) and 100 (ARR, 0.01) patients, respectively. Conclusions and Relevance In this analysis, findings suggest that for patients 60 years and older with hypertension, intensive BP treatment may be appropriate for some adults with a life expectancy of greater than 3 years but may not be suitable for those with less than 1 year

Time‐weighted blood pressure with cardiovascular risk among patients with or without diabetes

Background: Usual measures of blood pressure (BP) do not account for both the magnitude and duration of exposure to elevated BP over time. We aimed to demonstrate the effect of a novel time‐weighted BP on cardiovascular outcomes using a post hoc analysis of two published randomized trials. Hypothesis: Time‐weighted blood pressure is associated with cardiovascular risk among patients with or without diabetes. Methods: The limited‐access ACCORD and SPRINT data sets were used for the current study. Time‐weighted BP is obtained by dividing cumulative BP by the total follow‐up time. Time‐weighted BP burden above a threshold is also determined after deriving the time‐weighted BP by re‐zeroing the interpolated pressure values at two different hypertension thresholds (>140/90 and >130/80 mmHg). Results: Eighteen thousand five hundred forty‐one patients from the two clinical trials were enrolled in this study. A J‐curve relation was observed between time‐weighted BP and major cardiovascular events (MACE). The systolic blood pressure (SBP) burden independently predicted MACE across the two trials at different thresholds (ACCORD: SBP > 130 mmHg, HR = 1.05 [1.03−1.06]; SBP > 140 mmHg, HR = 1.06 [1.04−1.08]; SPRINT: SBP > 130 mmHg, HR = 1.04 [1.03−1.05]; SBP > 140 mmHg, HR = 1.05 [1.04−1.07]). Consistent results were found for diastolic blood pressure (DBP) burden (ACCORD: DBP > 80 mmHg, HR = 1.10 [1.06−1.15]; DBP > 90 mmHg, HR = 1.20 [1.11−1.30]. SPRINT: DBP > 80 mmHg, HR = 1.06 [1.02−1.09]; DBP > 90 mmHg, HR = 1.12 [1.06−1.18]). Significant associations were also observed for stroke, myocardial infarction, cardiovascular death, and all‐cause mortality. Conclusion: Both time‐weighted SBP and DBP independently influenced the risk of adverse cardiovascular events among patients with and without diabetes, regardless of the definition of hypertension (130/80 or <140/90 mmHg)

Genotyping and biofilm formation of Mycoplasma hyopneumoniae and their association with virulence

Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson’s index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae

Comparative Genomics of Mycoplasma: Analysis of Conserved Essential Genes and Diversity of the Pan-Genome

Mycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on 3,355 homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. There appears to be an approximate correlation between the divergence of species and the level of positive selection detected in corresponding lineages

Engineering new functions and altering existing functions

Structural and mechanistic information, sequence comparisons, and site-directed mutagenesis data continue to provide a basis for the rational design of new protein functions and the alteration of existing functions. Random mutagenesis and ‘directed evolution’ approaches, however, are making significant headway in solving protein engineering problems, proving highly practical for tuning properties such as enzyme substrate specificity