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Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Patient-reported outcomes (PROs) are increasingly recognized as valuable sources of information to enhance our understanding of the quality of healthcare from the patient's perspective., Objective: This study aimed to describe the implementation process of the Danish nationwide PRO-Psychiatry project, including iterative tests of previously developed PRO measurement concept and an online data collection tool. Additional aims were to identify the 'best practice' for the routine use of PROs in hospital-based psychiatry and design information material about the project., Methods: We conducted an action-oriented observational study to explore the pilot implementation of the PRO-Psychiatry project, which was initiated in February 2018. The study was based on an iterative plan-do-learn approach. An inpatient unit and an outpatient unit from the same psychiatric department in the North Denmark Region were selected for the pilot implementation. The implementation was anchored in multidisciplinary implementation teams at unit level. These teams managed the implementation process according to four tasks defined by the department management., Results: The teams designed, tested, evaluated and adjusted the localized work practices relating to the use of PRO-Psychiatry. The comprehensibility of the predesigned PROs, the usability of the Information Technology(IT) system and the routine use of PROs during clinical consultations were repeatedly tested and adjusted until the functionality was satisfactory. Furthermore, the teams designed information material for patients (emails, posters, handouts and webpages) and clinicians (online clinical guidelines). The team members informed their colleagues about the progress of PRO-Psychiatry at staff meetings and rolled out the initiative through one-to-one teaching., Conclusions: The pilot implementation was deemed successful. PRO-Psychiatry was rolled out to other units in the region, and a national decision was made to pilot implement the initiative in the other four Danish regions., (© The Author(s) 2021. Published by Oxford University Press on behalf of International Society for Quality in Health Care. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Reporting of barriers and successes associated with the implementation and use of patient-reported outcomes (PROs) is limited as a means to ensure enhanced patient involvement, shared decision-making and improved treatment and care. We set out to evaluate the implementation and use of the PRO-Psychiatry initiative on patient-reported outcome measures in Danish mental health care. We aimed to described four specific areas: the quality of the clinical consultations before and after the implementation of PRO-Psychiatry as perceived by the patients (objective A), the motivation for participating in PRO-Psychiatry as perceived by patients and clinicians (objective B), the implementation process as perceived by patients, clinicians and managers (objective C) and suggestions for improvement (objective D)., Methods: The PRO-Psychiatry initiative was evaluated through a participatory approach, including patients, clinicians and managers. A repeated cross-sectional interview-based survey explored the quality of the clinical consultation before and after the implementation of PRO-Psychiatry. A three-step semi-structured group interview, inspired by the modified mini-Delphi method, was used to establish consensus on the evaluation of the implementation and use of the initiative., Results: The evaluation pointed at PRO-Psychiatry as a meaningful initiative, which motivated patients and supported clinicians. The patients emphasised the importance of PROs, but they also found that PROs were not used enough. Clinically relevant improvements were detected after the implementation of the initiative; more patients felt heard and experienced that clinicians took a greater interest in their problems. The clinicians valued the easily accessible real-time graphical display of the PRO responses in the electronic health record (EHR). Clinicians and managers agreed that clinical PRO practices, patient compliance and use of PROs in treatment and care should be supported during implementation., Conclusion: The evaluation was overall positive. Patients and clinicians were willing to participate, found the online reporting easy and valued the direct access to PRO responses in the EHR. An essential feature was the integration of well-defined and functional PRO practices into the existing clinical workflow. Using PROs in the clinical sessions in a way that was palpable to the patient was found to be a significant improvement need. At the individual level, PRO-Psychiatry can use patient outcome information to support dialogue, encourage shared decision-making and promote self-management during recovery. At the aggregated patient level, the PROs can be used for monitoring the patient-perceived quality of care and for research., (© The Author(s) 2022. Published by Oxford University Press on behalf of International Society for Quality in Health Care. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The antipsychotic drug thioridazine is a candidate drug for an alternative treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in combination with the β-lactam antibiotic oxacillin. The drug has been shown to have the capability to resensitize MRSA to oxacillin. We have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall and affect the ability of the bacteria to sustain oxacillin treatment. Furthermore, we found that thioridazine itself reduces the expression level of selected virulence genes and that selected toxin genes are not induced by thioridazine. In the present study, we find indications that the mechanism underlying reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We have developed a two-site ELISA for measurement in serum of bone-related degradation products derived from C-terminal telopeptides of type I collagen. The assay is based on the application of two highly specific monoclonal antibodies against the amino acid sequence of AHD-beta-GGR, where the aspartic acid residue (D) is beta-isomerized. In a one-step incubation procedure, the degradation products containing cross-linked diisomerized EKAHD-beta-GGR peptides are captured by a biotinylated antibody and a peroxidase-conjugated antibody. The generated complex is then bound to the streptavidin surface via the biotin conjugate. Desalted urinary antigens are used for standardization, and parallelism is observed with serum samples. Results are obtained in <2.5 h, and both inter- and intraassay imprecision are <8%. The serum CrossLaps concentration was 1748+/-740 pmol/L (mean +/- SD) in premenopausal women (n = 65) and 2952+/-1325 pmol/L in a group of healthy postmenopausal women (n = 169). The Serum CrossLaps One Step ELISA was capable of detecting a highly significant (P <0.001) effect of hormone replacement therapy in a retrospective study involving 22 postmenopausal women.

We present a coated-tube RIA that is useful for assessment of bone resorption. The assay uses a monoclonal antibody raised against a linear 8-amino-acid sequence (EKAHDGGR) derived from the C-telopeptides of type I collagen. Within-run and total CVs were 4.4% and 5.3-6.2%, respectively, at concentrations of 1-7 mg/L (n = 4-20). Analytical recovery was 98% +/- 8% and dilution 97% +/- 7%. Values obtained in a group of 36 premenopausal women were 227 +/- 89.6 mg/mol creatinine. In a group of 141 postmenopausal women, the values obtained were 429 +/- 225 mg/mol creatinine, a highly significant increase of 89% (P <0.001) over the premenopausal value. In a double-blind placebo-controlled clinical study of these postmenopausal women receiving five different doses of a bisphosphonate, a significant decrease of RIA-measured C-telopeptide values was seen in all bisphosphonate-treated groups, after just 3 months. Values in urine samples from postmenopausal women assayed with the RIA (gamma) and the CrossLaps(TM) ELISA (x) agreed well: slope = 0.98 (95% confidence interval, 0.94-1.01), intercept = 0.34 (0.25-0.43) mg/L, and Sylx = 0.93 mg/L (n = 678). We conclude that this RIA represents a valuable tool for assessing bone resorption.

An enzyme-linked immunosorbent immunoassay (ELISA) for a new marker of bone resorption (CrossLaps) was evaluated. The ELISA procedure determines degradation products of type I collagen in urine. Values obtained in the ELISA and in pyridinoline by high pressure liquid chromatography were correlated after a correction for creatinine. A high correlation was found (r = 0.77; n = 81). A group of postmenopausal women (n = 180) showed an increase of more than 70% compared to values in premenopausal women (n = 104). Hydroxyproline was increased by 23%, osteocalcin by 52%, pyridinoline by 31%, and deoxypyridinoline by 50%. A highly significant decrease (60.7%) in the CrossLaps values was seen after 12 months in samples from patients receiving hormone replacement therapy compared to a placebo group. The spontaneous bone loss in an untreated group of women was determined by repeated forearm bone mass measurement over 24 months. Baseline values obtained in the CrossLaps ELISA were correlated to the rate of loss, yielding a highly significant r value of -0.61, indicating that CrossLaps might be a useful parameter for assessment of the risk of osteoporosis in postmenopausal women.

An enzyme-linked immunosorbent assay (ELISA) for measuring type I collagen degradation products in urine < 3 h was evaluated. The measuring range was 0.5-10.5 mg/L with a detection limit of 0.2 mg/L. Within-run and total CVs were 5.3% and 6.6%, respectively. Analytical recovery averaged 100%. The mean (+/- SD) concentrations in urine samples from a healthy premenopausal population (n = 102) were 250 +/- 110 mg/mol creatinine (Cr). A group of healthy postmenopausal women (n = 410) gave a mean value of 416 +/- 189 mg/mol Cr. Values obtained in the ELISA correlated well (r = 0.83) to HPLC values for the established bone resorption marker deoxypyridinoline (n = 214), slightly better than the correlation to hydroxyproline measurements (r = 0.78, n = 421). We conclude that the assay described here presents a useful tool for further elucidating the importance of type I collagen degradation products in urine.

A heterologous ELISA for measurement of osteocalcin (bone Gla-protein) is described, involving biotinylated bovine osteocalcin and polyclonal antibodies. The log-linear range was 2.3-37.5 micrograms/L. Between-run (total) and within-run CVs (n = 10) were 5.7-6.4 and 5.9-6.1%, respectively; analytical recoveries ranged from 92% to 108%. Comparison of our method (x) with an RIA (y) yielded y = 1.10 x -0.01 microgram/L, Sylx = 1.4 micrograms/L, r = 0.958 (n = 167). Serum osteocalcin in healthy premenopausal women (n = 29) was 8.7 +/- 3.3 micrograms/L (mean +/- SD) and 11.8 +/- 4.5 micrograms/L in early-postmenopausal women (n = 24). The assay was evaluated in a double-blind placebo-controlled study of healthy early-postmenopausal women, treated for 12 months with either (a) estrogen valerate plus medroxyprogesterone acetate (n = 18), (b) 17 beta-estradiol and desogestrel (n = 22), or (c) placebo (n = 17). Serum osteocalcin decreased significantly (P < 0.001) with either therapy, but increased (P < 0.05) with placebo.

We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).