The rate at which mutations are generated is central to the pace of evolution. Although this rate is remarkably similar amongst all cellular organisms, bacterial strains with mutation rates 100 fold ...greater than the modal rates of their species are commonly isolated from natural sources and emerge in experimental populations. Theoretical studies postulate and empirical studies teort the hypotheses that these "mutator" strains evolved in response to selection for elevated rates of generation of inherited variation that enable bacteria to adapt to novel and/or rapidly changing environments. Less clear are the conditions under which selection will favor reductions in mutation rates. Declines in rates of mutation for established populations of mutator bacteria are not anticipated if such changes are attributed to the costs of augmented rates of generation of deleterious mutations. Here we report experimental evidence of evolution towards reduced mutation rates in a clinical isolate of Escherichia coli with an hyper-mutable phenotype due a deletion in a mismatch repair gene, (ΔmutS). The emergence in a ΔmutS background of variants with mutation rates approaching those of the normal rates of strains carrying wild-type MutS was associated with increase in fitness with respect to ancestral strain. We postulate that such an increase in fitness could be attributed to the emergence of mechanisms driving a permanent "aerobic style of life", the negative consequence of this behavior being regulated by the evolution of mechanisms protecting the cell against increased endogenous oxidative radicals involved in DNA damage, and thus reducing mutation rate. Gene expression assays and full sequencing of evolved mutator and normo-mutable variants supports the hypothesis. In conclusion, we postulate that the observed reductions in mutation rate are coincidental to, rather than, the selective force responsible for this evolution.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CTX-M β-lactamases are the most prevalent group of enzymes within the extended-spectrum β-lactamases (ESBL). The therapeutic options for CTX-M-carrying isolates are scarce, forcing the reexamination ...of the therapeutic possibilities of β-lactams plus β-lactamase inhibitors (BBLIs). Inhibitor-resistant CTX-M β-lactamases (IR-CTX-M) have not hitherto been described in natural isolates. In this study, 168 cultures of the hypermutagenic Escherichia coli GB20 strain carrying plasmid pBGS18 with different blaCTX-M genes were submitted to parallel experimental evolution assays in the presence of increasing concentrations of a combination of amoxicillin and clavulanate. Fourteen CTX-M β-lactamases belonging to the three most representative clusters (CTX-M-1, -2, and -9) and the two main phenotypes (cefotaxime resistance and cefotaxime-ceftazidime resistance) were studied. Three types of IR-CTX-M mutants were detected, having mutations S130G, K234R, and S237G, which are associated with different resistance patterns. The most frequently recovered mutation was S130G, which conferred the highest resistance levels to BBLIs (reaching 12 µg/ml for amoxicillin-clavulanate and 96 µg/ml for piperacillin-tazobactam when acquired by CTX-M-1 cluster enzymes). The S130G change also provided a clear antagonistic pleiotropy effect, strongly decreasing the enzyme's activity against all cephalosporins tested. A double mutation, S130G L169S, partially restored the resistance against cephalosporins. A complex pattern observed in CTX-M-58, carrying P167S and S130G or K234R changes, conferred ESBL and IR phenotypes simultaneously. The K234R and S237G changes had a smaller effect in providing inhibitor resistance. In summary, IR-CTX-M enzymes might evolve under exposure to BBLIs, and the probability is higher for enzymes belonging to the CTX-M-1 cluster. However, this process could be delayed by antagonistic pleiotropy.
•Rapid detection of HBV helps guide treatment and assesses risk of complications.•Alinity m HBV assay runs on a fully automated, continuous, random-access platform.•Assay performance was examined at ...9 independent sites in serum and plasma samples.•Alinity m HBV assay had high correlation with 4 other assays across HBV genotypes.•Alinity m HBV assay showed linear quantitation, high precision, and reproducibility.
Accurate molecular methods to detect and quantify hepatitis B virus (HBV) DNA are essential to diagnose chronic infections, guide treatment decisions, assess response to treatment, and determine risk of HBV-related complications. New generations of real-time HBV DNA assay platforms provide results in less than 2−3 h, with continuous loading of specimens and true random-access capability.
We examined the clinical performance of the new Alinity m HBV assay, run on the fully automated, continuous, random-access Alinity m platform, to accurately detect and quantify HBV DNA in a large series of patient samples infected with different HBV genotypes frequently encountered in clinical practice.
This international, multisite study assessed the precision and reproducibility of the Alinity m HBV assay and compared its performance to four HBV assays currently in clinical use.
The Alinity m HBV assay demonstrated linear quantitation of HBV DNA in plasma samples, with high precision (coefficient of variation 4.1 %–8.8 %) and reproducibility. The Alinity m HBV assay showed excellent correlation (correlation coefficients ≥0.947) with comparator HBV assays, with an overall observed bias ranging from −0.07 to 0.17 Log10 IU/mL. 97 % of quantifiable patient results were <1 Log10 IU/mL different than the respective comparator assays, with comparable results across HBV genotypes.
The newly developed real-time PCR-based Alinity m HBV assay is sensitive, reproducible, and accurately quantifies HBV DNA levels from HBsAg-positive patients across the full dynamic range of quantification.
While most patients affected by the influenza A(H1N1) pandemic experienced mild symptoms, a small fraction required hospitalization, often without concomitant factors that could explain such a severe ...course. We hypothesize that host genetic factors could contribute to aggravate the disease. To test this hypothesis, we compared the allele frequencies of 547,296 genome-wide single nucleotide polymorphisms (SNPs) between 49 severe and 107 mild confirmed influenza A cases, as well as against a general population sample of 549 individuals. When comparing severe vs. mild influenza A cases, only one SNP was close to the conventional p = 5×10-8. This SNP, rs28454025, sits in an intron of the GSK233 gene, which is involved in a neural development, but seems not to have any connections with immunological or inflammatory functions. Indirectly, a previous association reported with CD55 was replicated. Although sample sizes are low, we show that the statistical power in our design was sufficient to detect highly-penetrant, quasi-Mendelian genetic factors. Hence, and assuming that rs28454025 is likely to be a false positive, no major genetic factor was detected that could explain poor influenza A course.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Imipenem/relebactam is a novel carbapenem/β-lactamase inhibitor combination, developed to act against carbapenemase-producing Enterobacterales (CPE).
Objectives
To assess the in ...vitro activity of imipenem/relebactam against a Spanish nationwide collection of CPE by testing the susceptibility of these isolates to 16 widely used antimicrobials and to determine the underlying β-lactam resistance mechanisms involved and the molecular epidemiology of carbapenemases in Spain.
Materials and methods
Clinical CPE isolates (n = 401) collected for 2 months from 24 hospitals in Spain were tested. MIC50, MIC90 and susceptibility/resistance rates were interpreted in accordance with the EUCAST guidelines. β-Lactam resistance mechanisms and molecular epidemiology were characterized by WGS.
Results
For all isolates, high rates of susceptibility to colistin (86.5%; MIC50/90 = 0.12/8 mg/L), imipenem/relebactam (85.8%; MIC50/90 = 0.5/4 mg/L) and ceftazidime/avibactam (83.8%, MIC50/90 = 1/≥256 mg/L) were observed. The subgroups of isolates producing OXA-48-like (n = 305, 75.1%) and KPC-like enzymes (n = 44, 10.8%) were highly susceptible to ceftazidime/avibactam (97.7%, MIC50/90 = 1/2 mg/L) and imipenem/relebactam (100.0%, MIC50/90 = ≤0.25/1 mg/L), respectively.
The most widely disseminated high-risk clones of carbapenemase-producing Klebsiella pneumoniae across Spain were found to be ST11, ST147, ST392 and ST15 (mostly associated with OXA-48) and ST258/512 (in all cases producing KPC).
Conclusions
Imipenem/relebactam, colistin and ceftazidime/avibactam were the most active antimicrobials against all CPEs. Imipenem/relebactam is a valuable addition to the antimicrobial arsenal used in the fight against CPE, particularly against KPC-producing isolates, which in all cases were susceptible to this combination.
The epidemiology of sexually transmitted infections (STIs) is complex due to the coexistence of various pathogens, the variety of transmission modes derived from sexual orientations and behaviors at ...different ages and genders, and sexual contact hotspots resulting in network transmission. There is also a growing proportion of recreational drug users engaged in high-risk sexual activities, as well as pharmacological self-protection routines fostering non-condom practices. The frequency of asymptomatic patients makes it difficult to develop a comprehensive approach to STI epidemiology. Modeling approaches are required to deal with such complexity. Membrane computing is a natural computing methodology for the virtual reproduction of epidemics under the influence of deterministic and stochastic events with an unprecedented level of granularity. The application of the LOIMOS program to STI epidemiology illustrates the possibility of using it to shape appropriate interventions. Under the conditions of our basic landscape, including sexual hotspots of individuals with various risk behaviors, an increase in condom use reduces STIs in a larger proportion of heterosexuals than in same-gender sexual contacts and is much more efficient for reducing
than
and lymphogranuloma venereum infections. Amelioration from diagnostic STI screening could be instrumental in reducing
infections, particularly in men having sex with men (MSM), and
infections in the heterosexual population; however, screening was less effective in decreasing lymphogranuloma venereum infections in MSM. The influence of STI epidemiology of sexual contacts between different age groups (<35 and ≥35 years) and in bisexual populations was also submitted for simulation.IMPORTANCEThe epidemiology of sexually transmitted infections (STIs) is complex and significantly influences sexual and reproductive health worldwide. Gender, age, sexual orientation, sexual behavior (including recreational drug use and physical and pharmacological protection practices), the structure of sexual contact networks, and the limited application or efficiency of diagnostic screening procedures create variable landscapes in different countries. Modeling techniques are required to deal with such complexity. We propose the use of a simulation technology based on membrane computing, mimicking in silico STI epidemics under various local conditions with an unprecedented level of detail. This approach allows us to evaluate the relative weight of the various epidemic drivers in various populations at risk and the possible outcomes of interventions in particular epidemiological landscapes.
Although the anti-HER2 antibody trastuzumab augments patient survival in HER2+ breast cancer, a relevant number of patients progress to this treatment. In this context, novel drug combinations are ...needed to increase its antitumor activity. In this work, we have evaluated the efficacy of proteolysis targeting chimera (PROTAC) compounds based on BET inhibitors (BETi) to augment the activity of trastuzumab in HER2+ breast cancer models.
BT474 and SKBR3 HER2+ breast cancer cell lines were used. The effects of trastuzumab and the BET-PROTAC MZ1 either alone or in combination, were evaluated using MTT proliferation assays, three-dimensional invasion and adhesion cultures, flow cytometry, qPCR and Western blot. In vivo studies were carried out in a xenografted model in mice. Finally, a Clariom_S_Human transcriptomic array was applied to identify deregulated genes after treatments.
MZ1 induced a higher antiproliferative effect compared to the BETi JQ1. The combination of MZ1 and -trastuzumab significantly decreased cell proliferation, the formation of three-dimensional structures and cellular invasion compared to either of the drugs alone. Evaluation of apoptosis resulted in an increase of cell death following treatment with the combination, and biochemical studies displayed modifications of apoptosis and DNA damage components. In vivo administration of agents alone or combined, to tumors orthotopically xenografted in mice, resulted in a decrease of the tumor volume only after MZ1-Trastuzumab combination treatment. Results from a transcriptomic array indicated a series of newly described transcription factors including HOXB7, MEIS2, TCERG1, and DNAJC2, that were associated to poor outcome in HER2+ breast cancer subtype and downregulated by the MZ1-trastuzumab combination.
We describe an active novel combination that includes the BET-PROTAC MZ1 and trastuzumab, in HER2+ tumors. Further studies should be performed to confirm these findings and pave the way for their future clinical development.
BackgroundTesting used in screening, diagnosis and follow-up of COVID-19 has been a subject of debate. Several organisations have developed formal advice about testing for COVID-19 to assist in the ...control of the disease. We collated, delineated and appraised current worldwide recommendations about the role and applications of tests to control SARS-CoV-2/COVID-19.MethodsWe searched for documents providing recommendations for COVID-19 testing in PubMed, EMBASE, LILACS, the Coronavirus Open Access Project living evidence database and relevant websites such as TRIP database, ECRI Guidelines Trust, the GIN database, from inception to 21 September 2020. Two reviewers applied the eligibility criteria to potentially relevant citations without language or geographical restrictions. We extracted data in duplicate, including assessment of methodological quality using the Appraisal of Guidelines for Research and Evaluation-II tool.ResultsWe included 47 relevant documents and 327 recommendations about testing. Regarding the quality of the documents, we found that the domains with the lowest scores were ‘Editorial independence’ (Median=4%) and ‘Applicability’ (Median=6%). Only six documents obtained at least 50% score for the ‘Rigour of development’ domain. An important number of recommendations focused on the diagnosis of suspected cases (48%) and deisolation measures (11%). The most frequently recommended test was the reverse transcription-PCR (RT-PCR) assay (87 recommendations) and the chest CT (38 recommendations). There were 22 areas of agreement among guidance developers, including the use of RT-PCR for SARS-Cov-2 confirmation, the limited role of bronchoscopy, the use chest CT and chest X-rays for grading severity and the co-assessment for other respiratory pathogens.ConclusionThis first scoping review of recommendations for COVID-19 testing showed many limitations in the methodological quality of included guidance documents that could affect the confidence of clinicians in their implementation. Future guidance documents should incorporate a minimum set of key methodological characteristics to enhance their applicability for decision making.
The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new ...molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials.
Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs.
In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.
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•CASCADE is a novel detection system that combines Cas13a and gold nanoparticles.•It enables fast and colorimetric detection of SARS-CoV-2 RNA.•Coupled to isothermal amplification, it can detect SARS-CoV-2 in clinical samples.•CASCADE is a good candidate for fast naked-eye detection in point-of-care diagnosis.
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