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.

Continuity and discontinuity of care among older patients in Danish general practice: a retrospective cohort study.

BACKGROUND: Continuity of care (COC) for older adults has been associated with lower use of healthcare services, decreased risk of hospitalisation, and lower mortality. However, research on COC in older adults is limited by short time periods and small sample sizes. Long-term COC can only develop if the patient stays with the general practice for ≥10 years. Therefore, research that focuses on long duration and broader populations is needed. AIM: To measure the extent of longitudinal site-level COC in general practice and listing duration of the patient-general practice relation for all older Danish citizens. DESIGN & SETTING: Retrospective cohort study of all patients aged ≥65 years on 31 December 2021 listed with a Danish general practice (N = 1 144 941 persons). METHOD: Individual-level register data were used on start and end dates for listing with a general practice to analyse site-level COC by number of changes and listing duration of the patient-general practice relation from January 2007-December 2021. RESULTS: During the 15 years, 39.3% of older adults did not change general practice. Among the remaining 60.7%, who experienced discontinuity of care, 34.0% changed once, 16.3% changed twice, and 6.3% changed three times. Overall, <5% changed general practice >3 times. The duration of the patient-general practice relations were on average 9.5 years. Overall, 27.5% lasted 0-4 years, 33.7% lasted 5-9 years, and 38.8% lasted ≥10 years. CONCLUSION: Danish general practice provides high levels of site-level COC for their older patients. On average, patients aged ≥65 years changed general practice once and had a patient-general practice relation length of 9.5 years.

Variation in daytime general practice services and association with out-of-hours use for older patients: protocol for a Danish cohort study.

INTRODUCTION: Out-of-hours primary care services cannot provide the same continuity and coordination of care as general practice. Thus, patients with high risk of complex care trajectories should, when possible, be treated by the general practitioner during daytime opening hours. This study aims to analyse the variation among general practices in the frequencies of daytime services for persons aged ≥75 years and how it relates to the patients' use of out-of-hours services. METHODS AND ANALYSIS: Register-based cohort study of all Danish citizens aged ≥75 years, of whom >98% are listed with a general practice. Using Poisson regression, we will estimate each practice's excess variation in delivered daytime services compared with the expected based on the characteristics of its listed patients. Delivered daytime services will be analysed overall and separately for face-to-face, phone, email, home visit and preventive services. The association with the use of out-of-hours services will be analysed by Poisson regression. ETHICS AND DISSEMINATION: Complying with European data protection rules, the legal services at University of Southern Denmark (Research & Innovation Organisation) approved the data processing activities regarding this project (journal number 11.593). According to section 14.2 of the Act on Research Ethics Review of Health Research Projects, because the study is based solely on register data, approval from the ethics committee and informed consent are not required. Results from the study will be disseminated as publications in peer-reviewed scientific journals and at international conferences.

Optical coherence tomography quantifying photo aging: skin microvasculature depth, epidermal thickness and UV exposure.

INTRODUCTION: Photo aging predominantly occurs in the face, neck and hands due to UVA and UVB irradiation. It is associated with skin cancer and histological studies indicate thinning of the epidermis and elastosis occurs. Dynamic Optical coherence tomography (D-OCT) is a non-invasive imaging tool able to visualize the epidermis and upper dermis and its blood vessels as well as to evaluate epidermal thickness (ET) and blood flow. OBJECTIVE: To investigate ET and blood vessel depth using D-OCT in human subjects correlated to UV exposure. METHODS: We evaluated data from 249 healthy adults, that had D-OCT-scans conducted at four different regions (forehead, neck, arm and hand) and correlated ET and blood vessel depth with occupational UV exposure (total standard erythema dose, Total SED), season and demographic data. RESULTS: Regional differences in ET and blood vessel depth were found (p values < 0.001). Multiple linear regressions showed a seasonal effect on both ET (- 0.113 to - 0.288 µm/day, p values < 0.001) and blood vessel depth (0.168-0.347 µm/day, p values < 0.001-0.007) during August-December. Significant age-related decrease of ET was seen in forehead, arm and hand (0.207-0.328 µm/year, p values = 0.002-0.18) and blood vessel depth in forehead (0.064-0.553 µm/year, p values = 0.01-0.61). Males had thicker epidermis (3.92-10.93 µm, p values = 0.002-0.15). CONCLUSION: Changing seasons are a major predictor of both ET and blood vessel depth, showing strongest effect in non-exposed areas, suggesting a systemic effect, possibly due to seasonal vitamin D fluctuation. Sex, age and occupational UV exposure affect ET. This study demonstrated the feasibility of D-OCT to evaluate epidermal thickness and blood vessel depth.

C-reactive protein cut-offs used for acute respiratory infections in Danish general practice.

BACKGROUND: GPs can use the C-reactive protein (CRP) point-of-care test (POCT) to assist when deciding whether to prescribe antibiotics for patients with acute respiratory tract infections (RTIs). AIM: To estimate the CRP cut-off levels that Danish GPs use to guide antibiotic prescribing for patients presenting with different signs and symptoms of RTIs. DESIGN & SETTING: A cross-sectional study conducted in general practice in Denmark. METHOD: During the winters of 2017 and 2018, 143 GPs and their staff registered consecutive patients with symptoms of an RTI according to the Audit Project Odense (APO) method. CRP cut-offs were estimated as the lowest level at which half of the patients were prescribed an antibiotic. RESULTS: In total, 7813 patients were diagnosed with an RTI, of whom 4617 (59%) had a CRP test performed. At least 25% of the patients were prescribed an antibiotic when the CRP level was >20 mg/L, at least 50% when CRP was >40 mg/L, and at least 75% when CRP was >50 mg/L. Lower thresholds were identified for patients aged ≥65 years and those presenting with a fever, poor general appearance, dyspnoea, abnormal lung auscultation, or ear/facial pain, and if the duration of symptoms was either short (≤1 day) or long (>14 days). CONCLUSION: More than half of patients presenting to Danish general practice with symptoms of an RTI have a CRP test performed. At CRP levels >40 mg/L, the majority of patients have an antibiotic prescribed.

Optical coherence tomography of onychomycosis: proposed terminology and a suggestion of practical usage.

Onychomycosis is the most common cause of nail changes accounting for about half of all nail diseases. It is important to diagnose the fungal pathogen to prescribe adequate treatment, but as genus- and species-specific diagnostic tests can be time consuming and expensive, it is crucial to obtain representative tissue during nail scrapings. Optical coherence tomography (OCT) is a non-invasive imaging tool enabling real-time imaging of skin and tissue. Pilot studies using OCT have described morphological characteristics of onychomycosis, though diagnostic criteria of onychomycosis and other nail diseases are lacking. The aim of this study is to investigate the applicability of OCT to detect and localize fungal elements in nails with clinical signs of onychomycosis. OCT scans and nail scrapings were performed on all participants and only confirmed cases of onychomycosis were included in the data analysis. All scans were analyzed, and a set of morphological criteria based on known literature and deviations from healthy nail morphology were suggested. The suggested morphological features were: hyperreflective lines divided into sharply and diffusely demarcated; hyperreflective dots divided into singular and clustered patterns; irregular surface, divided into mild, moderate and severe degree; dark bands and disturbed architecture. The suggested OCT morphology is a refinement of previous studies, and though OCT cannot replace genus- or species-specific identification, it could be a useful assistance tool to increase diagnostic sensitivity of nail scrapings and laboratory tests as well as for monitoring treatment response.

Advances in optical coherence tomography in dermatology-a review.

Optical coherence tomography (OCT) was introduced as an imaging system, but like ultrasonography, other measures, such as blood perfusion and polarization of light, have enabled the technology to approach clinical utility. This review aims at providing an overview of the advances in clinical research based on the improving technical aspects. OCT provides cross-sectional and en face images down to skin depths of 0.4 to 2.00 mm with optical resolution of 3 to 15 µm. Dynamic optical coherence tomography (D-OCT) enables the visualization of cutaneous microvasculature via detection of rapid changes in the interferometric signal of blood flow. Nonmelanoma skin cancer (NMSC) is the most comprehensively investigated topic, resulting in improved descriptions of morphological features and diagnostic criteria. A refined scoring system for diagnosing NMSC, taking findings from conventional and D-OCT into account, is warranted. OCT diagnosis of melanoma is hampered by the resolution and the optical properties of melanin. D-OCT may be of value in diseases characterized with dynamic changes in the vasculature of the skin and the addition of functional measures is strongly encouraged. In conclusion, OCT in dermatology is still an emerging technology that has great potential for improving further in the future.

Dermatobia hominis misdiagnosed as abscesses in a traveler returning from Brazil to Denmark.

We present the case of a 62-year-old woman that consulted us for two boil-like lesions on her thighs after returning from a trip to São Paulo, Brazil, where she had swum in a freshwater lake. After consulting three specialist doctors and undergoing two antibiotic treatments, she was diagnosed with furuncular myiasis caused by Dermatobia hominis. The parasites were excised with no complications.