Physical and psychological status of emergency assistance personnel at major public health events: a qualitative descriptive study.

BACKGROUND: Many personnel respond to natural disasters like earthquakes and tsunamis and frequent public health events like Ebola and COVID-19. However, research on emergency assistance personnel remains limited. This study aims to describe the perceived well-being among responders deployed in isolated or emergency international missions while providing practical references to intervene in developing similar missions. METHODS: For this qualitative phenomenological study, purposive sampling was used following the principle of maximum differentiation to select personnel deployed on an emergency mission for over a year. Data collection continued until data saturation. Phenomenologically semi-structured interviews helped explore the physical and psychological status of the participants with Colaizzi's method. RESULTS: Eleven personnel were interviewed after the mission, with four major themes being identified: 'perceived somatic change,' 'perceived emotional change,' 'behavioral change,' and 'coping with perceived change.' CONCLUSIONS: The mental health status of the emergency assistance personnel was affected by multiple factors from external and internal environments. The current study explored the physical and psychological feelings and emotions of emergency assistance personnel during an emergency mission. The study provided a practical reference for health management under similar missions. REGISTRATIONS: Not registered.

Echocardiographic assessment of pediatric heart transplantation: A single-center experience in China.

BACKGROUND: Pediatric heart transplant (HT) has become the standard of care for end-stage heart failure in children worldwide. Serial echocardiographic evaluations of graft anatomy and function during follow-up are crucial for post-HT management. However, evolution of cardiac structure and function after pediatric HT has not been well described, especially during first year post-HT. This study aimed to characterize the evolution of cardiac structure and function after pediatric HT and investigate the correlation between biventricular function with adverse clinical outcomes. METHODS: A single-center retrospective study of echocardiographic data obtained among 99 pediatric HT patients was conducted. Comprehensive echocardiographic examination was performed in all patients at 1-, 3-, 6-, 9- and 12-months post-HT. We obtained structural, functional and hemodynamic parameters from both left- and right-side heart, such as left ventricular stroke volume (LVSV), left ventricular ejection fraction (LVEF), right ventricular fractional area change (RVFAC), etc. The cardiac evolution of pediatric HT patients during first post-HT year was described and compared between different time points. We also explored the correlation between cardiac function and major adverse transplant events (MATEs). RESULTS: 1) Evolution of left heart parameters: left atrial length, mitral E velocity, E/A ratio, LVSV and LVEF significantly increased while mitral A velocity significantly decreased over the first year after HT (P < .05). Compared with 1 month after HT, interventricular septum (IVS) and left ventricular posterior wall (LVPW) decreased at 3 months but increased afterwards. (2) Evolution of right heart parameters: right ventricular base diameter and mid-diameter; right ventricular length diameter, tricuspid E velocity, E/A ratio, tricuspid annular velocity e' at free wall, and RVFAC increased, while tricuspid A velocity decreased over the first year after HT (P < .05). (3) Univariate logistic regression model suggests that biventricular function parameters at 1-year post-HT (LVEF, RVFAC, tricuspid annular plane systolic excursion and tricuspid lateral annular systolic velocity) were associated with MATEs. CONCLUSION: Gradual improvement of LV and RV function was seen in pediatric HT patients within the first year. Biventricular function parameters associated with MATEs. The results of this study pave way for designing larger and longer follow-up of this population, potentially aiming at using multiparameter echocardiographic prediction of adverse events.

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

Uncertainty quantification of PM2.5 concentrations using a hybrid model based on characteristic decomposition and fuzzy granulation.

The prediction of air pollution plays an important role in reducing the emission of air pollutants and guiding people to carry out early warning and control, so it attracts many scholars to conduct modeling and research on it. However, most of the current researches fail to quantify the uncertainty in prediction and only use traditional fuzzy information granulation to process data, resulting in the loss of much detail information. Therefore, this paper proposes a hybrid model based on decomposition and granular fuzzy information to solve these problems. The trend item and the Granulation fluctuation item are respectively predicted and the results are combined to obtain the change trend and fluctuation range of the sequence. This paper selects PM2.5 concentrations of 3 cities. The experimental results show that the evaluation index of the prediction model is significantly lower than other benchmark models, and a variety of statistical methods are used to further verify the effectiveness of the prediction model.

The mismatch repair system (mutS and mutL) in Acinetobacter baylyi ADP1.

BACKGROUND: Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi. RESULTS: We determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (RifR assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from RifR assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in RifR assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in RifR assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background. CONCLUSIONS: We demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor.

BACKGROUND: Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. RESULTS: In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. CONCLUSION: These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.

Dual Role of gnaA in Antibiotic Resistance and Virulence in Acinetobacter baumannii.

Acinetobacter baumannii is an important Gram-negative pathogen in hospital-related infections. However, treatment options for A. baumannii infections have become limited due to multidrug resistance. Bacterial virulence is often associated with capsule genes found in the K locus, many of which are essential for biosynthesis of the bacterial envelope. However, the roles of other genes in the K locus remain largely unknown. From an in vitro evolution experiment, we obtained an isolate of the virulent and multidrug-resistant A. baumannii strain MDR-ZJ06, called MDR-ZJ06M, which has an insertion by the ISAba16 transposon in gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase), a gene found in the K locus. The isolate showed an increased resistance toward tigecycline, whereas the MIC decreased in the case of carbapenems, cephalosporins, colistin, and minocycline. By using knockout and complementation experiments, we demonstrated that gnaA is important for the synthesis of lipooligosaccharide and capsular polysaccharide and that disruption of the gene affects the morphology, drug susceptibility, and virulence of the pathogen.

Double-Diamond Model-Based Orientation Guidance in Wearable Human-Machine Navigation Systems for Blind and Visually Impaired People.

This paper presents the analysis and design of a new, wearable orientation guidance device in modern travel aid systems for blind and visually impaired people. The four-stage double-diamond design model was applied in the design process to achieve human-centric innovation and to ensure technical feasibility and economic viability. Consequently, a sliding tactile feedback wristband was designed and prototyped. Furthermore, a Bezier curve-based adaptive path planner is proposed to guarantee collision-free planned motion. Proof-of-concept experiments on both virtual and real-world scenarios are conducted. The evaluation results confirmed the efficiency and feasibility of the design and imply the design's remarkable potential in spatial perception rehabilitation.

Comparison of RECIST, EORTC criteria and PERCIST for evaluation of early response to chemotherapy in patients with non-small-cell lung cancer.

PURPOSE: To compare the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, the European Organization for Research and Treatment of Cancer (EORTC) criteria and the Positron Emission Tomography Response Criteria in Solid Tumors (PERCIST) 1.0 using PET volume computer-assisted reading (PET VCAR) for response evaluation in patients with advanced non-small-cell lung cancer (NSCLC) treated with chemotherapy. METHODS: A total of 35 patients with NSCLC were included in this prospective study. All patients received standard chemotherapy and underwent (18)F-FDG PET/CT scans before and after treatment. With the assistance of PET VCAR, the chemotherapeutic responses were evaluated according to the RECIST 1.1, EORTC criteria and PERCIST 1.0. Concordance among these protocols was assessed using Cohen's κ coefficient and Wilcoxon's signed-ranks test. Progression-free survival (PFS) was calculated using the Kaplan-Meier test. RESULTS: RECIST 1.1 and EORTC response classifications were discordant in 20 patients (57.1 %; κ = 0.194, P < 0.05), and RECIST 1.1 and PERCIST 1.0 classifications were discordant in 22 patients (62.9 %; κ = 0.139, P < 0.05). EORTC and PERCIST 1.0 classifications were discordant in only 4 patients (11.4 %), resulting in better concordance (κ = 0.804, P > 0.05). Patients with a partial remission according to RECIST 1.1 had significantly longer PFS (P < 0.001) than patients with progressive disease, but not significantly longer than patients with stable disease (P = 0.855). According to both the EORTC criteria and PERCIST 1.0, patients with a partial metabolic response had a significantly longer PFS than those with stable metabolic disease and those with progressive metabolic disease (P = 0.020 and P < 0.001, respectively, for EORTC; both P < 0.001 for PERCIST 1.0). CONCLUSION: EORTC criteria and PERCIST 1.0 are more sensitive and accurate than RECIST 1.1 for the detection of an early therapeutic response to chemotherapy in patients with NSCLC. Although EORTC criteria and PERCIST 1.0 showed similar results, PERCIST 1.0 is preferred because detailed and unambiguous definitions are given. We also found that response evaluations with PERCIST 1.0 using a single lesion and multiple lesions gave similar response classifications.

Transesophageal echocardiography related complications.

Transesophageal Echocardiography (TEE) is an important imaging method for the evaluation of cardiac structure and function, and it holds significant value in the clinical management of cardiovascular diseases. Unlike transthoracic echocardiography (TTE), which is non-invasive, TEE involves semi-invasive intracavity operations, leading to increasing attention to its safety and potential complications. Especially with the increasing demand for TEE applications in clinical practice and the rapid growth in the number of facilities utilizing it, the standardized application and safe operation of TEE technology have become particularly crucial. This article will review the literature and draw upon personal experience to analyze the complications and safety of TEE examinations from a technical perspective.

Research of a combination system based on fuzzy sets and multi-objective marine predator algorithm for point and interval prediction of wind speed.

Short-term wind speed forecasting is fundamental to improving the stability of power grid operation and enhancing its transmission efficiency; thus, it has long been a research hotspot. Nonetheless, quantities of literature in this field only used the single prediction model and overemphasized deterministic prediction, which resulted in deficient forecasting performance. To address these issues, a novel point and interval combination prediction system was developed in this paper. Specifically, wind speed time series were reconstructed by dividing windows and fuzzification to input highly effective data; next, four single prediction models and a multi-objective weight-determining mechanism were integrated to obtain the point prediction results; and their distributions were assessed to implement interval prediction under distinct confidence levels. In the meantime, this study demonstrated that the proposed system reached the Pareto optimal by the theoretical proof, and empirical research was conducted based on 10-min real wind speed data from the wind farm in China. Judging from the experimental results, the combination prediction system was always capable of providing the most satisfactory forecasting performance by contrast with the comparative models. Consequently, it has broad application prospects in guiding the operation of wind farms and optimizing the power grid dispatching.

Mutation in the two-component regulator BaeSR mediates cefiderocol resistance and enhances virulence in Acinetobacter baumannii.

Acinetobacter baumannii has become one of the most challenging pathogens in many countries with limited treatment options available. Cefiderocol, a novel siderophore-conjugated cephalosporin, shows potent in vitro activity against A. baumannii, including isolates resistant to carbapenems. To date, few reports on the mechanisms of cefiderocol resistance are available. In order to investigate potential mechanisms of cefiderocol resistance in A. baumannii, we performed in vitro evolution experiments at sub-lethal concentrations of the antibiotic. All four cefiderocol-resistant strains obtained harbored mutations in two-component system BaeS-BaeR. When we engineered the mutations of BaeS (D89V) and BaeR (S104N) into the genome of ATCC 17978, these mutations increased cefiderocol minimum inhibitory concentrations (MICs) by 8-fold to 16-fold. Transcriptome analyses showed that the expression of MacAB-TolC and MFS transporters was up-regulated in BaeSR mutants. Strains over-expressing MFS transporter and MacAB-TolC displayed higher MICs and higher median inhibition concentration (IC50) values, while MICs and IC50 decreased when efflux pump genes were knocked out. In a BaeR mutant with up-regulated csu operon, we observed a higher number of pili, enhanced surface motility, and increased biofilm formation compared to wild-type ATCC 17978. Using the Galleria mellonella infection model, we found that the BaeS mutant in which paa operon was up-regulated exhibited increased virulence. In conclusion, the mutations in BaeSR decreased cefiderocol susceptibility of A. baumannii through up-regulating efflux pumps gene expression. BaeS or BaeR also controls the expression of csu and paa, influencing biofilm formation, surface motility, and virulence in A. baumannii. IMPORTANCE The widespread prevalence of multi-drug-resistant A. baumannii (MDRAB) poses a significant therapeutic challenge. Cefiderocol is considered a promising antibiotic for the treatment of MDRAB infections. Therefore, it is necessary to study the potential resistance mechanisms of cefiderocol to delay the development of bacterial resistance. Here, we demonstrated that mutations in baeS and baeR reduced the susceptibility of A. baumannii to cefiderocol by up-regulating the expression of the MFS family efflux pump and MacAB-TolC efflux pump. We propose that BaeS mutants increase bacterial virulence by up-regulating the expression of the paa operon. This also reports the regulatory effect of BaeSR on csu operon for the first time. This study provides further insights into the role of BaeSR in developing cefiderocol resistance and virulence in A. baumannii.

Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

Noncanonical cofactors such as nicotinamide mononucleotide (NMN+) supplant the electron-transfer functionality of the natural cofactors, NAD(P)+, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden-Meyerhof-Parnas (EMP) glycolytic pathway to generate NMN+-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydrogenase (Sm GapN) to utilize NMN+. Through iterative rounds of rational design, we discover the variant GapN Penta (P179K-F153S-S330R-I234E-G210Q) with high NMN+-dependent activity and GapN Ortho (P179K-F153S-S330R-I234E-G214E) with ~3.4 × 106-fold switch in cofactor specificity from its native cofactor NADP+ to NMN+. GapN Ortho is further demonstrated to function in Escherichia coli only in the presence of NMN+, enabling orthogonal control of glucose utilization. Molecular dynamics simulation and residue network connectivity analysis indicate that mutations altering cofactor specificity must be coordinated to maintain the appropriate degree of backbone flexibility to position the catalytic cysteine. These results provide a strategy to guide future designs of NMN+-dependent enzymes and establish the initial steps toward an orthogonal EMP pathway with biomanufacturing potential.

Co-evolutionary adaptations of Acinetobacter baumannii and a clinical carbapenemase-encoding plasmid during carbapenem exposure.

OXA-23 is the predominant carbapenemase in carbapenem-resistant Acinetobacter baumannii. The co-evolutionary dynamics of A. baumannii and OXA-23-encoding plasmids are poorly understood. Here, we transformed A. baumannii ATCC 17978 with pAZJ221, a bla OXA-23-containing plasmid from clinical A. baumannii isolate A221, and subjected the transformant to experimental evolution in the presence of a sub-inhibitory concentration of imipenem for nearly 400 generations. We used population sequencing to track genetic changes at six time points and evaluated phenotypic changes. Increased fitness of evolving populations, temporary duplication of bla OXA-23 in pAZJ221, interfering allele dynamics, and chromosomal locus-level parallelism were observed. To characterize genotype-to-phenotype associations, we focused on six mutations in parallel targets predicted to affect small RNAs and a cyclic dimeric (3' → 5') GMP-metabolizing protein. Six isogenic mutants with or without pAZJ221 were engineered to test for the effects of these mutations on fitness costs and plasmid kinetics, and the evolved plasmid containing two copies of bla OXA-23 was transferred to ancestral ATCC 17978. Five of the six mutations contributed to improved fitness in the presence of pAZJ221 under imipenem pressure, and all but one of them impaired plasmid conjugation ability. The duplication of bla OXA-23 increased host fitness under carbapenem pressure but imposed a burden on the host in antibiotic-free media relative to the ancestral pAZJ221. Overall, our study provides a framework for the co-evolution of A. baumannii and a clinical bla OXA-23-containing plasmid in the presence of imipenem, involving early bla OXA-23 duplication followed by chromosomal adaptations that improved the fitness of plasmid-carrying cells.

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform.

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.

Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst.

Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor stability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate an aerobic, high-throughput growth selection platform in Escherichia coli for oxygenase evolution based on NADH redox balance. We applied this NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We first identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a ∼1200-fold relative cofactor specificity switch from NADPH to NADH compared to the wild type through semirational design. Molecular modeling suggests CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Additional evolution of CHMO DTNP through random mutagenesis yielded the variant CHMO DTNPY with a ∼2900-fold relative specificity switch compared to the wild type afforded by an additional distal mutation, H163Y. These results highlight the difficulty in engineering functionally innovative variants from static models and rational designs, and the need for high throughput selection methods. Our introduced tools for oxygenase engineering accelerate the advancements of characteristics essential for industrial feasibility.

Novel tigecycline resistance mechanisms in Acinetobacter baumannii mediated by mutations in adeS, rpoB and rrf.

Acinetobacter baumannii is an important pathogen in hospital acquired infections. Although tigecycline currently remains a potent antibiotic for treating infections caused by multidrug resistant A. baumannii (MDRAB) strains, reports of tigecycline resistant isolates have substantially increased. The resistance mechanisms to tigecycline in A. baumannii are far more complicated and diverse than what has been described in the literature so far. Here, we characterize in vitro-selected MDRAB strains obtained by increasing concentrations of tigecycline. We have identified mutations in adeS, rrf and rpoB that result in reduced susceptibility to tigecycline. Using in situ complementation experiments, we confirm that mutations in rrf, rpoB, and two types of mutations in adeS correlate with tigecycline resistance. By Western blot and polysome profile analysis, we demonstrate that the rrf mutation results in decreased expression of RRF, which affects the process of ribosome recycling ultimately leading to increased tigecycline tolerance. A transcriptional analysis shows that the mutated rpoB gene plays a role in regulating the expression of the SAM-dependent methyltransferase (trm) and transcriptional regulators, to confer moderate tigecycline resistance. This study provides direct in vitro evidence that mutations in the adeS, rpoB and rrf are associated with tigecycline resistance in A. baumannii.

A random forest model based on core genome allelic profiles of MRSA for penicillin plus potassium clavulanate susceptibility prediction.

Treatment failure of methicillin-resistant Staphylococcus aureus (MRSA) infections remains problematic in clinical practice because therapeutic options are limited. Penicillin plus potassium clavulanate combination (PENC) was shown to have potential for treating some MRSA infections. We investigated the susceptibility of MRSA isolates and constructed a drug susceptibility prediction model for the phenotype of the PENC. We determined the minimum inhibitory concentration of PENC for MRSA (n=284) in a teaching hospital (SRRSH-MRSA). PENC susceptibility genotypes were analysed using a published genotyping scheme based on the mecA sequence. mecA expression in MRSA isolates was analysed by qPCR. We established a random forest model for predicting PENC-susceptible phenotypes using core genome allelic profiles from cgMLST analysis. We identified S2-R isolates with susceptible mecA genotypes but PENC-resistant phenotypes; these isolates expressed mecA at higher levels than did S2 MRSA (2.61 vs 0.98, P<0.05), indicating the limitation of using a single factor for predicting drug susceptibility. Using the data of selected UK-sourced MRSA (n=74) and MRSA collected in a previous national survey (NA-MRSA, n=471) as a training set, we built a model with accuracies of 0.94 and 0.93 for SRRSH-MRSA and UK-sourced MRSA (n=287, NAM-MRSA) validation sets. The AUROC of this model for SRRSH-MRSA and NAM-MRSA was 0.96 and 0.97. Although the source of the training set data affects the scope of application of the prediction model, our data demonstrated the power of the machine learning approach in predicting susceptibility from cgMLST results.

In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO).

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.

A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site.

We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and we demonstrate its application in the high-throughput evolution of an oxygenase. A single round of selection followed by a facile growth assay enabled Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA), two consecutive steps in gallic acid biosynthesis. Structural modeling suggests precise reorganization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.