Investigating Protein Binding with the Isothermal Ligand-induced Resolubilization Assay.

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand-induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand-induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate-linked moiety X motif 5 (NUDT5), poly [ADP-ribose] polymerase 1 (PARP1), and protein arginine N-methyltransferase 1 (PRMT1). Ligand-induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand-induced protein solubility were assessed by Coomassie blue staining for SDS-PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.

The durability of previous examinations for cancer: Danish nationwide cohort study.

OBJECTIVE: Patients previously examined for cancer with a negative result may present in general practice with ongoing or new symptoms or signs suggestive of cancer. This paper explores the potential existence of a relatively safe period for cancer occurrence after receiving negative examination results for specific types of cancer, including lung (CT thorax), upper gastrointestinal (gastroscopy), colorectal (colonoscopy), bladder (cystoscopy), and breast (clinical mammography). DESIGN: Register-based time-to-event analyses. SETTING: Denmark. SUBJECTS: All 3.3 million citizens aged 30-85 years who on January first, 2017, had not previously been diagnosed with the specific type of cancer were categorized based on the time since their most recent examination. MAIN OUTCOME MEASURES: Using 1-year follow-up, we calculated the age- and sex-adjusted hazard ratios of being diagnosed with the related cancer, with non-examined individuals as reference. Negative examination results were defined as the absence of a cancer diagnosis within 6 months following the examination. RESULTS: Previous negative examination results were common, also among those diagnosed with cancer during follow-up. For 10 years after a negative colonoscopy the risk of diagnosing a colorectal cancer was nearly halved. However, already 1 year after a clinical mammography and 2 years after a CT thorax the risk of diagnosing the related cancers was significantly higher among those with a previous negative result compared to non-examined individuals. CONCLUSION: This study did not identify a post-examination period in which the cancer risk, compared to non-examined individuals, was sufficiently low to confidently rule out any of the investigated cancers.

What should one expect when considering re-examining a patient with a negative result of a previous examination for cancer? We found that previous negative examination results are common in the general population and among those subsequently diagnosed with cancer. We did not find a safe period after any of the examinations in which a negative result alone could safely rule out the presence of cancer.

Attempted treatment of primary axillary hyperhidrosis with one session of either Neodymium YAG laser or Intense Pulsed Light: A within patient randomized trial of treated versus untreated contralateral axilla.

BACKGROUND: Primary axillary hyperhidrosis (PAH) affects 1-5% of the world's population who has an unmet need for improved treatments. The heating of sweat glands with specific microwave therapy has shown promising results, yet, treatment with widely available devices such as long-pulsed Neodymium Yttrium Aluminum Garnet (Nd:YAG) lasers, diode lasers or Intense Pulsed Light (IPL) may serve as pragmatic alternatives. OBJECTIVES: To compare sweat secretion of treated versus untreated contralateral control axilla 1-3 months after one session of Nd:YAG laser or IPL in patients with PAH. METHODS: A within-person randomized controlled trial. Patients were randomized to receive either one session of Nd:YAG laser or IPL in one axilla with the contra-lateral serving as control. Sweat production was assessed by gravimetry, trans-epidermal water loss, hyperhidrosis disease severity scale and dynamic optical coherence tomography. Mixed-effects models were used to handle the within-person design, containing both fixed effect factors (side, group, and subgroup), and random effects (patients), while also adjusting for the level at baseline. RESULTS: A total of 20 patients were enrolled. At follow-up 1-3 months after treatment, sweat secretion was not affected in the treated axilla when compared to the control axillae (0.01 [95%CI: -0.04 to 0.05]; p = 0.68). In the Nd:YAG subgroup (10 patients), least squares means for sweat secretion was 0.18 mg/5 min in the treated versus 0.15 mg/5 min in the control axilla, respectively, corresponding to a statistically insignificant mean difference of 0.02 mg/5 min (95% CI: -0.06 to 0.11; p = 0.54). In the IPL subgroup (10 patients), sweat secretion was 0.06 mg/5 min in the treated axilla versus 0.07 mg/5 min in the control axilla with a statistically insignificant difference of -0.01 points (95% CI: -0.03 to 0.02; p = 0.46). Likewise, none of the secondary outcomes were significantly affected by treatment. However, both treatments appeared safe and well tolerated with no adverse effects reported at follow-up. CONCLUSIONS: One treatment with external 1064 nm Nd:YAG laser or 640 nm IPL at commercially available settings, failed to demonstrate clinical benefit in treating PAH, with narrow confidence intervals implying that this was not due to a type-2 error.

Dynamic Optical Coherence Tomography Imaging of Telangiectasia Prior to Intense Pulsed Light Treatment-An Opportunity to Target Treatment?

BACKGROUND AND OBJECTIVES: To investigate whether optical coherence tomography (OCT) could be utilized to characterize blood flow and vessel dimensions of facial telangiectasias before and during consecutive intense pulsed light (IPL) treatment. STUDY DESIGN/MATERIALS AND METHODS: Dynamic OCT (D-OCT) was used to image telangiectasia immediately before and after, 1-3 days after, and 1 month after IPL treatment. Measurements included vessel width and depth, blood flow, and attenuation. Vessel dimensions at baseline were verified by a blinded observer. Clinical improvement was detected as good, moderate, or none, and adverse effects were registered at 1-month follow-up. RESULTS: In total, 14 patients with facial telangiectasia were included. At baseline, vessel width was median 0.25 mm (interquartile range [IQR]: 0.19-0.34 mm) with an intra-class coefficient (ICC) of 0.89 (95% confidence interval [CI]: 0.70; 0.97). Vessel depth was 0.30 mm (IQR: 0.25-0.33 mm; ICC: 0.40 [CI: -0.07; 0.75]). Vessel depth increased significantly from baseline to 1-month follow-up (P = 0.008), whereas no significant changes in vessel width, blood flow, or attenuation were detected. Clinical efficacy seemed related to the relation between vessel dimensions and applied energy settings. CONCLUSIONS: The D-OCT imaging technique demonstrated that facial telangiectasias were found deeper within the skin after one IPL treatment. By characterizing the vessel dimensions and blood flow of telangiectasia, D-OCT may improve efficacy and safety of IPL. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Prescription of antibiotics to children with acute otitis media in Danish general practice.

BACKGROUND: Acute otitis media (AOM) is a common and most often self-limiting infection in childhood, usually managed in general practice. Even though antibiotics are only recommended when certain diagnostic and clinical criteria are met a high antibiotic prescription rate is observed. The study's objective was to analyse associations between patient- and general practitioner (GP) characteristics and antibiotic prescribing for children with AOM in an effort to explain the high antibiotic prescribing rates. METHODS: All general practices in the Northern, Southern and Central regions of Denmark were invited to record symptoms, examinations, findings and antibiotic treatment for all children ≤7 years of age diagnosed with AOM during a four-week winter period in 2017/2018. Associations were analysed by means of multivariate logistic regressions. The study design was cross-sectional. RESULTS: GPs from 60 general practices diagnosed 278 children with AOM of whom 207 (74%) were prescribed antibiotics, most often penicillin V (60%). About half of the children had tympanometry performed. Antibiotic prescribing rates varied considerably between practices (0-100%). Antibiotic prescribing was associated with fever (odds ratio (OR) 3.69 95% confidence interval (CI) 1.93-7.05), purulent ear secretion (OR 2.35 95% CI 1.01-5.50) and poor general condition (OR 3.12 95% CI 1.31-7.46), and the practice's antibiotic prescribing rate to other patients with symptoms of an acute respiratory tract infection (OR 2.85 CI 95% 1.07-7.60) and specifically to other children with AOM (OR 4.15 CI 95% 1.82-9.47). CONCLUSION: GPs' antibiotic prescribing rates for children with AOM vary considerably even considering the of signs, symptoms, request for antibiotics, and use of tympanometry. Interventions to reduce overprescribing should be targeted high-prescribing practices.

Photoaging and actinic keratosis in Danish outdoor and indoor workers.

BACKGROUND: The risk of photoaging and actinic keratosis caused by work related solar ultraviolet radiation exposure has not previously been investigated in Nordic countries. The objectives of this study were to describe the occurrence of photoaging, actinic keratosis, and keratinocyte cancer in a population of Danish outdoor and indoor workers, and investigate the association between these clinical findings and semi-objective measures of work related solar ultraviolet radiation exposure in the same population. METHODS: A clinical cross-sectional study of the occurrence of facial wrinkles, actinic keratosis, keratinocyte cancer, and melanocytic nevi in a population of Danish outdoor and indoor workers and associations with semi-objective measures of work related solar ultraviolet radiation exposure based on a combination of dosimetry and self-report. RESULTS: Work related solar ultraviolet radiation exposure was significantly positively associated with occurrence of facial wrinkles (α = 0.05). Actinic keratosis was associated to status as outdoor worker (OR = 4.272, CI [1.045-17.471]) and age (P < 0.001, CI [1.077-1.262]) and twice as common in outdoor workers (10.3% CI [0.05, 0.15]) compared to indoor workers (5.1% CI [0.00, 0.10]). Only two cases of keratinocyte cancer were diagnosed (<1%). Older age was negatively associated with occurrence of melanocytic nevi. CONCLUSION: Outdoor work in Denmark is associated with increased occurrence of facial wrinkles and actinic keratosis from solar ultraviolet radiation exposure, thus justifying sun safety at Danish workplaces from a clinical perspective.

Vascular morphology in normal skin studied with dynamic optical coherence tomography.

Dynamic optical coherence tomography (D-OCT) is a non-invasive imaging technique, suitable for the study of structural and dynamic features of cutaneous microvasculature. Studies with D-OCT have primarily focused on non-melanoma skin cancer (NMSC), and a reference description of healthy skin is lacking. The aim of the study was to describe the prevalence of standard microvascular features in normal skin. A total of 280 participants without skin disease were D-OCT-scanned on four body locations: three sun-exposed areas and one unexposed: forehead, back of the neck, back of the hand and medial side of the upper arm. Frequencies of standard vascular features were reported, and relations to anatomical location and demographic data were investigated. "Dots," "lines" and "curves" were the most frequent shapes at 150 µm, 300 µm and 500 µm. "Mottle" was the predominant pattern at 150 µm and 300 µm. "Mesh" was found from 300 µm and primarily found at 500 µm. Regional differences in vascular characteristics were primarily found comparing the medial side of the arm with the other body locations. In normal skin, the most frequent shapes were "dots," "lines" and "curves," and "mottle" was present more superficially than "mesh." In conclusion, regional anatomical differences should be taken into account when evaluating D-OCT images.

In vivo differentiation of common basal cell carcinoma subtypes by microvascular and structural imaging using dynamic optical coherence tomography.

The subtype of basal cell carcinoma (BCC) influences the choice of treatment. Optical coherence tomography (OCT) is a non-invasive imaging tool, and a recent development of an angiographic version of OCT has extended the application of OCT to image the cutaneous microvasculature (so-called dynamic OCT, D-OCT). This study explores D-OCT's ability to differentiate the common BCC subtypes by microvascular and structural imaging. Eighty-one patients with 98 BCC lesions, consisting of three subtypes: 27 superficial BCC (sBCC), 55 nodular BCC (nBCC) and 16 infiltrative BCC (iBCC) were D-OCT scanned at three European dermatology centres. Blinded evaluations of microvascular and structural features were performed, followed by extensive statistical analysis of risk ratio (RR) and multiple correspondence analysis. nBCC lesions displayed most characteristic structural and vascular features. Serpiginous vessels, branching vessels, vessels creating a circumscribed figure and sharply demarcated hyporeflective ovoid structures in the dermis were all associated with a higher risk of the subtype being nBCC. The presence of highly present lines and dark peripheral borders at the margin of ovoid structures was negatively associated with iBCC. Lastly, the finding of hyporeflective ovoid structures protruding from epidermis correlated with sBCC. We identified various microvascular and structural D-OCT features that may aid non-invasive identification of BCC subtypes. This would allow clinicians to individualize and optimize BCC treatment as well as aid follow-up of non-surgical treatment.

Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens.

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Improving Antibiotic Use in Nursing Homes by Infection Prevention and Control and Antibiotic Stewardship (IMAGINE): Protocol for a Before-and-After Intervention and Implementation Study.

BACKGROUND: Despite the extensive use of antibiotics and the growing challenge of antimicrobial resistance, there has been a lack of substantial initiatives aimed at diminishing the prevalence of infections in nursing homes and enhancing the detection of urinary tract infections (UTIs). OBJECTIVE: This study aims to systematize and enhance efforts to prevent health care-associated infections, mainly UTIs and reduce antibiotic inappropriateness by implementing a multifaceted intervention targeting health care professionals in nursing homes. METHODS: A before-and-after intervention study carried out in a minimum of 10 nursing homes in each of the 8 European participating countries (Denmark, Greece, Hungary, Lithuania, Poland, Slovakia, Slovenia, and Spain). A team of 4 professionals consisting of nurses, doctors, health care assistants, or health care helpers are actively involved in each nursing home. Over the initial 3-month period, professionals in each nursing home are registering information on UTIs as well as infection and prevention control measures by means of the Audit Project Odense method. The audit will be repeated after implementing a multifaceted intervention. The intervention will consist of feedback and discussion of the results from the first registration, training on the implementation of infection and prevention control techniques provided by experts, appropriateness of the diagnostic approach and antibiotic prescribing for UTIs, and provision of information materials on infection control and antimicrobial stewardship targeted to staff, residents, and relatives. We will compare the pre- and postintervention audit results using chi-square test for prescription appropriateness and Student t test for implemented hygiene elements. RESULTS: A total of 109 nursing homes have participated in the pilot study and the first registration audit. The results of the first audit registration are expected to be published in autumn of 2024. The final results will be published by the end of 2025. CONCLUSIONS: This is a European Union-funded project aimed at contributing to the battle against antimicrobial resistance through improvement of the quality of management of common infections based on evidence-based interventions tailored to the nursing home setting and a diverse range of professionals. We expect the intervention to result in a significant increase in the number of hygiene activities implemented by health care providers and residents. Additionally, we anticipate a marked reduction in the number of inappropriately managed UTIs, as well as a substantial decrease in the overall incidence of infections following the intervention. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/60099.