Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical ...transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.
IMPORTANCE: SARS-CoV-2 infection is associated with persistent, relapsing, or new symptoms or other health effects occurring after acute infection, termed postacute sequelae of SARS-CoV-2 infection ...(PASC), also known as long COVID. Characterizing PASC requires analysis of prospectively and uniformly collected data from diverse uninfected and infected individuals. OBJECTIVE: To develop a definition of PASC using self-reported symptoms and describe PASC frequencies across cohorts, vaccination status, and number of infections. DESIGN, SETTING, AND PARTICIPANTS: Prospective observational cohort study of adults with and without SARS-CoV-2 infection at 85 enrolling sites (hospitals, health centers, community organizations) located in 33 states plus Washington, DC, and Puerto Rico. Participants who were enrolled in the RECOVER adult cohort before April 10, 2023, completed a symptom survey 6 months or more after acute symptom onset or test date. Selection included population-based, volunteer, and convenience sampling. EXPOSURE: SARS-CoV-2 infection. MAIN OUTCOMES AND MEASURES: PASC and 44 participant-reported symptoms (with severity thresholds). RESULTS: A total of 9764 participants (89% SARS-CoV-2 infected; 71% female; 16% Hispanic/Latino; 15% non-Hispanic Black; median age, 47 years IQR, 35-60) met selection criteria. Adjusted odds ratios were 1.5 or greater (infected vs uninfected participants) for 37 symptoms. Symptoms contributing to PASC score included postexertional malaise, fatigue, brain fog, dizziness, gastrointestinal symptoms, palpitations, changes in sexual desire or capacity, loss of or change in smell or taste, thirst, chronic cough, chest pain, and abnormal movements. Among 2231 participants first infected on or after December 1, 2021, and enrolled within 30 days of infection, 224 (10% 95% CI, 8.8%-11%) were PASC positive at 6 months. CONCLUSIONS AND RELEVANCE: A definition of PASC was developed based on symptoms in a prospective cohort study. As a first step to providing a framework for other investigations, iterative refinement that further incorporates other clinical features is needed to support actionable definitions of PASC.
Dark adaptometry may be a useful diagnostic test and clinical trial endpoint for age-related maculopathy (ARM) because impaired night vision is a hallmark of early ARM. A novel dark adaptometer, the ...AdaptDx, was evaluated for the detection of ARM. The AdaptDx incorporates a 20-minute protocol optimized for the detection of ARM. ARM patients (
N
= 17) exhibited substantial dark adaptation impairment compared with normal adults (
N
= 17). The diagnostic sensitivity was 88% and the specificity was 100%. The diagnostic test characteristics of the AdaptDx are similar to previously reported studies using 60- to 120-minute protocols.
Partial fossil frond impressions of a Gleichenia-like fern are described and figured from specimens recovered from the early Miocene Landslip Hill silcrete deposit, Gore Lignite Measures, Southland, ...New Zealand. The fronds are at least bipinnate, with the pinnae (β costae) bearing opposite pairs of ultimate leaflets; ultimate segments opposite to alternate, laterally directed, ovate, slightly recurved, apices rounded obtuse. The sori are solitary per ultimate segment and there are 5-16 sporangial base scars per sorus. However, because of the difficulty of assigning Gleichenia-like partial fronds to genus, or even to Gleicheniaceae, the fossils are assigned to the fossil fern genus Korallipteris as a new species, K. alineae. Comparison with living and Cenozoic Gleichenia-like macrofossils suggests that the fossil shows morphological resemblance to the extant species G. inclusisora and G. microphylla. The macrofossils are discussed in terms of their possible palaeoenvironmental setting, together with abundant, potentially associated dispersed Gleicheniaceae fossil spores recovered from interbedded lignites. Assuming K. alineae is gleicheniaceous, it probably grew as a pioneer coloniser species in a periodically flooded/disturbed open deltaic riverine environment.
Objective Finite-element analysis demonstrates that the nonplanar shape of the mitral annulus diminishes mitral leaflet stress. It has therefore been postulated that repair with annuloplasty rings ...that maintain the nonplanar shape of the annulus could increase repair durability. Although the global nonplanarity of the mitral annulus has been adequately characterized, design of such a ring requires a quantitative description of regional annular geometry. By using real-time 3-dimensional echocardiography in conjunction with available image processing software, we developed a methodology for describing regional annular geometry and applied it to the characterization of the normal human mitral annulus. Methods Five healthy volunteers underwent mitral valve imaging with real-time 3-dimensional echocardiography. Regional annular height was calculated at 36 evenly spaced intervals. Results Maximal annular height/commissural width ratio was found to occur at the midpoint of the anterior annulus in all cases. These values averaged 26% ± 3.1%, whereas those for the midposterior annulus averaged 18% ± 3.0%. The average commissural width was 35.2 ± 6.0 mm. Although substantial spatial heterogeneity was observed, regional annular height at a given rotational position was highly conserved among subjects when normalized to commissural width. Conclusions These quantitative imaging and analytic techniques demonstrate that the normal human mitral annulus is regionally heterogeneous in its nonplanarity, and they establish a means of describing annular geometry at a regional level. With wider application, these techniques may be used both to characterize pathologic annular geometry and to optimize the design of mitral valve annuloplasty devices.
Study Design
Systemic review and meta-analysis.
Objective
To review and establish the effect of tobacco smoking on risk of nonunion following spinal fusion.
Methods
A systematic search of Medline, ...Embase, Cochrane Central Register of Controlled Trials, and the Cochrane Database of Systematic Reviews from inception to December 31, 2020, was conducted. Cohort studies directly comparing smokers with nonsmokers that provided the number of nonunions and fused segments were included. Following data extraction, the risk of bias was assessed using the Quality in Prognosis Studies Tool, and the strength of evidence for nonunion was evaluated using the GRADE working group criteria. All data analysis was performed in Review Manager 5, and a random effects model was used.
Results
Twenty studies assessing 3009 participants, which included 1117 (37%) smokers, met inclusion criteria. Pooled analysis found that smoking was associated with increased risk of nonunion compared to not smoking ≥1 year following spine surgery (RR 1.91, 95% CI 1.56 to 2.35). Smoking was significantly associated with increased nonunion in those receiving either allograft (RR 1.39, 95% CI 1.12 to 1.73) or autograft (RR 2.04, 95% CI 1.54 to 2.72). Both multilevel and single level fusions carried increased risk of nonunion in smokers (RR 2.30, 95% CI 1.64 to 3.23; RR 1.79, 95% CI 1.12 to 2.86, respectively).
Conclusion
Smoking status carried a global risk of nonunion for spinal fusion procedures regardless of follow-up time, location, number of segments fused, or grafting material. Further comparative studies with robust methodology are necessary to establish treatment guidelines tailored to smokers.
Abstract Individuals with schizophrenia demonstrate behavioral and neurobiological deficits in episodic memory. However, recent work suggests that episodic memory deficits in schizophrenia may be ...mitigated through specific encoding strategies. The current study directly compared brain activity and memory performance associated with two different verbal encoding orientations in the same group of schizophrenia participants, in order to more fully characterize the role of strategy in memory processing in this population. Participants included 18 individuals with schizophrenia and 15 healthy comparison participants. Participants encoded words under two conditions during separate fMRI scanning runs. During Incidental encoding, participants were required to make abstract/concrete judgments for each word. During Intentional encoding, participants were instructed to memorize each word for a later memory test. Free recall and a recognition task (utilizing the Remember/Know paradigm) were performed outside of the scanner. Consistent with prior work, schizophrenia participants recognized more words encoded Incidentally than Intentionally, although free recall remained substantially impaired. Schizophrenia participants were also less likely to give Remember judgments for old words and more likely to give Guess judgments for both old and new words. When functional magnetic resonance imaging data were examined, we found that Incidental encoding was associated with substantially fewer between-group differences (Control > Schizophrenia) than Intentional encoding. Furthermore, schizophrenia participants exhibited intact activity during encoding of items that were subsequently retrieved. Our results suggest that use of an Incidental encoding strategy improved recognition memory among individuals with schizophrenia and resulted in a pattern of encoding-related brain activity that was more similar to that seen in control participants. However, we found that Incidental encoding did not improve free recall in schizophrenia participants and abnormal brain activity in some regions was observed, despite improvements in recognition memory.
The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect ...societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats.
We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits.
These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation.
The purpose of this study was to assess the inflammatory nature of obesity and its effect on blood and bone marrow endothelial cell populations. Obese patients (BMI ≥30) had significantly higher ...concentrations of the inflammatory marker C‐reactive protein (CRP) (P = 0.03) and lower concentrations of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) (P = 0.05). This cytokine profile is consistent with obesity being an inflammatory condition and is further supported by the significant correlation between total white blood cell count and BMI (r = 0.15; P = 0.035). High BMI was associated with significantly lower numbers of early endothelial cells (CD45−/CD34+) in the bone marrow (r = −0.20; P = 0.0068). There was also a significant inverse correlation between BMI and a more mature endothelial cell phenotype (CD45−/31+) in the blood (r = −0.17; P = 0.02). In addition, there was a significant correlation between BMI‐ and endothelial‐related cells of hematopoietic origin (CD133+/VEGFR‐2+) in the bone marrow (r = −0.26; P = 0.0007). Patients with higher plasma IL‐10 and insulin‐like growth factor (IGF) concentrations had higher numbers of endothelial phenotypes in the bone marrow suggesting a protective effect of these anti‐inflammatory cytokines. In conclusion, this work confirms the inflammatory nature of obesity and is the first to report that obesity is associated with reduced endothelial cell numbers in the bone marrow of humans. These effects of obesity may be a potential mechanism for impaired tissue repair in obese patients.
Objective: To develop a tool to allow Australian hospitals to monitor the range of hospital‐acquired diagnoses coded in routine data in support of quality improvement efforts.
Design and setting: ...Secondary analysis of ed inpatient records for all episodes in acute care hospitals in Victoria for the financial year 2005–06 (n = 2.032 million) to develop a classification system for hospital‐acquired diagnoses; each record contains up to 40 diagnosis fields coded with the ICD‐10‐AM (International Classification of Diseases, 10th revision, Australian modification).
Main outcome measure: The Classification of Hospital Acquired Diagnoses (CHADx) was developed by: analysing codes with a “complications” flag to identify high‐volume code groups; assessing their salience through an iterative review by health information managers, patient safety researchers and clinicians; and developing principles to reduce double counting arising from coding standards.
Results: The dataset included 126 940 inpatient episodes with any hospital‐acquired diagnosis (complication rate, 6.25%). Records had a mean of three flagged diagnoses; including unflagged obstetric and neonatal codes, 514 371 diagnoses were available for analysis. Of these, 2.9% (14 898) were removed as comorbidities rather than complications, and another 118 640 were removed as redundant codes, leaving 380 833 diagnoses for grouping into CHADx classes. We used 4345 unique codes to characterise hospital‐acquired conditions; in the final CHADx these were grouped into 144 detailed subclasses and 17 “roll‐up” groups.
Conclusions: Monitoring quality improvement requires timely hospital‐onset data, regardless of causation or “preventability” of each complication. The CHADx uses routinely ed hospital diagnosis and condition‐onset information about in‐hospital complications. Use of this classification will allow hospitals to track monthly performance for any of the CHADx indicators, or to evaluate specific quality improvement projects.
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