Steel corrosion is major reason of the deterioration of reinforced concrete structures. Decreasing the transportation of erosion ions in concrete is one of effective methods to protect the steel from corrosion. In the present work, a novel nano-hydrophobic admixture is introduced to improve the ion-diffusion properties and the corrosion resistance of reinforced steel. Compared with unmodified concrete, the nano-hydrophobic admixture effectively decreases the water adsorption, water evaporation, and chloride ions transport in a concrete structure, and then improved the concrete’s durability. The concrete’s water adsorption decreased more than 78%, and the initial corrosion time of reinforced steel is prolonged more than one time by treatment with the nano-hydrophobic admixture. The inhibition penetration of the medium in concrete modified by hydrophobic nanoparticles is the key to provide the protective properties of steel reinforcement from chloride erosion. Concrete is the most widely used construction building material. It is widely used in civil engineering, such as housing, agriculture, roads, bridges, water conservancy facilities, underground structures, nuclear power plants, and marine development. As the global climate changes and the field of construction expands, especially for the harsh service environment, the deterioration of concrete structures becomes more and more serious, which brings the biggest challenge for sustainable social development in the future ,2,3. TheUsually, the steel in concrete is kept passivated in the high alkaline environment, which can protect the steel from corrosion by forming an oxide film. However, when the penetration of chloride ions reaches a certain concentration this will destroy the passive oxide film, or carbon dioxide diffusion into concrete will neutralize the alkalinity of the aqueous solution in the concrete pores leading to the instability of the passive oxide film . The staAt present, the improvement of the permeability of concrete is mainly achieved by improving the compactness of cement-based materials and optimizing the pore structure by reducing the water/cement ratio, and the addition of auxiliary cement-based materials or nanoparticles ,11,12,13For obtaining a more durable reinforced concrete structure, a nano-hydrophobic agent was incorporated into concrete. The effects of the nano-hydrophobic admixture on the corrosion behavior of reinforced steel and on concrete durability  were investigated. The nano-hydrophobic material is an aqueous emulsion, which can be easily dispersed in concrete. The purpose of this work is to explore the effect of a nano-hydrophobic admixture on the corrosion inhibition of steel in concrete, the resistance of water adsorption, and chloride ion transportation. The work will provide an experimental basis and application reference for the development of durable concrete.The steel used in the present work is HRB 400 reinforced steel, the composition of which is listed in Ordinary Portland cement PO 42.5 was provided by the Hailuo cement corporation. The chemical and mineralogical compositions of the cement are listed in 3, 15 kg/m3, 20 kg/m3, 25 kg/m3, and 30 kg/m3 by the volume of concrete were prepared. The mix proportions of the concrete are listed in The concrete with water-to-cement ratios (w/c) of 0.35 and nano-hydrophobic (TIA) admixture at dosages of 0, 10 kg/mThe reinforced steel concrete specimens were cast into prismatic specimens with the side lengths of 40 mm after 2 min mixing. The fresh concrete was cast into cubic specimens with side lengths of 150 mm after 2 min mixing. All samples were vibrated on the vibration table for 1 min before being sealed and then were stored under an environmental condition with a temperature of ca. 20 °C and a relative humidity above 95% for 1 day. Finally, all specimens were de-molded and cured under an environment with a temperature of ca. 20 °C and a relative humidity above 95% to the designed ages.For the reinforced steel corrosion test in concrete, prismatic specimens (40 × 40 × 100 mm in size) were produced . These pWater absorption tests were carried out using concrete specimens cured for 28 days, as according to BS 1881 part 122. For a representative sample, a set of three specimens was procured by obtaining a core 75 mm long when the thickness of the specimen was greater than 150 mm. The diameter of each core was 75 ± 3 mm. After drilling the core, the specimens were dried at 50 °C in an oven for 72 h; then each specimen was removed from the oven and cooled for 24 h in a dry airtight vessel. Each specimen was weighed and immediately completely immersed in the tank with its longitudinal axis horizontal and at a depth such that there was 25 mm of water covering it. The weight changes of the concrete were performed at 30 min, 60 min, 180 min, 300 min, 720 min, and 1440 min after the concrete began to contact the water. For each measurement, the specimen was quickly taken out, wiped with a dry towel to remove the free water from the surface, and then placed on the balance with the wet side up. The specimen should be put back immediately to continue the water-absorption test after weighing. The weighing process should be completed within 15 s. For each group of concrete, three specimens were tested and the average value was used as the experimental result.The specimens for the evaporation test are similar to those for the water adsorption test. The specimens were placed in an oven and refrigerator to keep the environment temperature at 50 °C, 25 °C, and 0 °C. The weight change with different times were recorded and calculated to determine the evaporation ratio.The chloride diffusion test was carried after dry–wet cycle experiment. The distribution of chloride ions from the depth of the concrete was detected by a chloride ion titration method, according to JGJ/T 322-2013. The samples, abrasive to powder, from different depths of concrete and immersions in acid solution to obtain the total chloride content. The chloride concentrations at different depths were recorded.Electrochemical measurement was undertaken to validate the nano-hydrophobic admixture on the corrosion behavior of reinforced steel and the distinction of the admixture on the influence of concrete penetration or direct reaction with steel surface. Corrosion was detected in both the steel embedded in concrete and that immersed in the simulated concrete-pore solution.The electrochemical corrosion behavior of the reinforced steel exposed to the chloride environment was evaluated by linear polarization measurements with respect to a saturated calomel electrode (SCE) as the reference and platinum as the counter-electrode. The electrochemical values reported in the graphs are averaged among the measurements carried out on three specimens of each concrete type during the immersion period.2, and a different concentration of TIA. The electrochemical parameter was detected after steel was immersed in the simulated solution for 24 h. This test was different from that of steel embedded in concrete because the steel electrode was directly in contact with chloride ions, and the kinetics of the steel corrosion process influenced by the nano-hydrophobic admixture was easily obtained. The electrochemical test for steel in the simulated concrete pore solution was followed by EIS measurements. The simulated pore solution was composed of 3.5 wt.% NaCl, saturated Ca(OH)The effects of immersion time and the content of nano-hydrophobic admixture on the water absorption of concrete were investigated; the results are shown in When TIA was added into concrete, the water adsorption was effectively reduced as the concentration of the admixture was increased, and the water absorption ratio decreases even more. During the test time, the water adsorption amount and water sorptivity of concrete were both reduced by TIA; the saturated adsorption process was prolonged more than in specimens without TIA. The most water adsorption ration reduced by TIA during stage I was about 78%, and at end of stage II, the TIA also decreased the water adsorption ration about 63%. The results show that the TIA has the most effective influence on the capillary adsorption process.For further investigation, the effect of TIA on the capillary adsorption process, different environment temperatures, and immersion time in the water adsorption of concrete by different dosages of TIA were explored . As the With the prolongation of soaking time, the overall change trend of water absorption of concrete with TIA concentration does not change. The difference is that at 0 °C, the higher mixing amount of TIA will lead to more reduction in water absorption; the result shows that the water absorption ratios all decrease with the concentration of TIA increasing at different environment temperatures, while the influence of temperature on water adsorption was less.The content of TIA has less effect on the evaporation of concrete during the initial exposure time a, while The influence of the exposure time on the water evaporation rate is also one of main factors in water evaporation. At the initial exposure time, the total evaporation amount is relatively less; as the time prolongs, the evaporation amount increases sharply for blank specimens. For concrete treated by a hydrophobic admixture, the evaporation ratio increases slowly, and more content of TIA also inhibits the water evaporation even more. After a longer time of exposure, the gap between the total amount of water evaporation becomes prominent, and the influence of the hydrophobic substance content on the water evaporation rate gradually increases. 3, the reinforcement is kept in the state of passivation after 10 cycles. The initial corrosion time of reinforced steel is prolonged more than one time. The corrosion of the reinforcement in concrete is mainly affected by the concentration of chloride ions on the surface of the reinforcement, the concentration of humidity and oxygen in the pore environment, and various substances in the pore solution ,6,7,8,9.As is well-known, water is the carrier of corrosive ions, and water adsorption is one form of driving ion transport. Normally, the water adsorption of concrete approximately follows the √t law ,29. Ther√t law . During √t law ,22,23,24γ is the surface tension, θ is the contact angle, and r is the capillary diameter.On the other hand, it is found that room temperature or a higher temperature has no effect on water absorption; this is mainly because the capillary pressure is the main driving force that impels water into concrete when the water was flowing as a liquid. As is well-known, the capillary pressure provided by a certain pore diameter r is shown in the follow formula :(1)P=2γcAt temperatures of 0~50 °C, the surface tension and contact angle of water change little in concrete; therefore, the temperature has less influence on the water absorption when the water is in a liquid and flowing condition. However, the pore distribution and pore size of concrete will change with the hydrophobic admixture ,12,13, wHowever, the water evaporation process was significantly influenced by the temperature; even the TIA dosage is a main influencing factor. Water evaporation can enhance the water adsorption and result in more of the chloride content gathering, which is the why the dry–wet cycle is the most serious cause of damage to ion transport and the deterioration of concrete ,18. At tThe above results show that TIA can effectively inhibit the water flow into and out of concrete. Therefore, the chloride ion transmission was effectively suppressed. Chloride ion transportation includes three forms: capillary transport, infiltration, and natural diffusion ,14. UndeBecause of water adsorption, water evaporation and chloride ion diffusion are restrained by TIA; therefore, the steel in concrete is effectively protected, but the steel surface is not changed by TIA. The inhibition of TIA on reinforced steel corrosion is mainly due to the improvement of the resistance of concrete medium permeability. For the future, the influence of TIA on the microstructures of concrete and the interface of steel and concrete should be study. Besides, the influence of this new hydrophobic agent on steel corrosion under other extreme service environments also should be paid attention to.(1)The nano-hydrophobic admixture exhibits a significant water adsorption inhibition behavior; the higher the content of TIA incorporated into concrete, the more water transportation-restraint performance was obtained. During the water adsorption process, the main influencing factor is the structure of the concrete pore modified by TIA, while the temperature has less effect on water transportation.(2)The nano-hydrophobic admixture also has an effective inhibition behavior on water evaporation, which is also influenced by environmental temperature. A higher content of TIA and a lower temperature favor reducing water evaporation.(3)Because the water adsorption and water evaporation process were both inhibited, the transportation of chloride ions was effectively restrained by the hydrophobic admixture. In the present investigation, the chloride ion concentration was reduced more than 50% compared with the blank concrete, which is normal marine engineering concrete.(4)The nano-hydrophobic admixture effectively prolongs steel corrosion by suppressing of chloride ion transport, which has less effect on electrochemical corrosion behavior when the chloride ions directly contact the steel surface.In this paper, the effects of a nano-hydrophobic admixture on concrete water adsorption, water evaporation, chloride ions transportation, and steel corrosion were investigated; the following conclusions can be drawn:


MET PET/CT scans were performed in nineteen patients before and 3 weeks after completion of CIRT. After Surgery, the effect of neoadjuvant CIRT was investigated by examining the relationship between each parameter of MET uptake and the histological assessment . Four parameters of MET uptake were the maximum and minimum standardized uptake values of pre and post CIRT . MET PET/CT imaging of esophageal cancer was clearly demonstrated. The post-SUVmax was the most suitable parameter. When the cutoff value was set as post-SUVmax = 6.21, the sensitivity, the specificity, and the accuracy of Grades 3 were 100.0%, 63.6%, and 78.9%, respectively. And there was a positive relationship between the tumor residual ratio and post-SUVmax . MET PET/CT is clinically useful for the assessment of early response to neoadjuvant CIRT in esophageal cancer. Particularly, post-SUVmax is considered a promising PET imaging parameter.This study aimed to evaluate the uptake of the clinical effectiveness of [S-methyl- The most typical curative treatment for esophageal cancer is surgery; however, in Japan, neoadjuvant chemotherapy is recommended to improve prognosis4. Regarding other neoadjuvant therapies, the efficacy of neoadjuvant chemoradiotherapy combined with radiation therapy has also been reported6.Esophageal cancer is the sixth leading cause of cancer mortality worldwide7. Carbon ion beams have a high relative biological effectiveness with a high linear energy transfer and are capable of creating a very high dose peak, which is called a Bragg peak. Considering these features, CIRT is more effective and causes less radiation damage to normal tissues than conventional radiotherapy. From 2004 to 2008, the world’s first phase I/II clinical trial of neoadjuvant CIRT for esophageal cancer was conducted in collaboration with the Department of Frontier Surgery, Graduate School of Medicine, Chiba University. CIRT has a significantly good therapeutic effect with a complete response (CR) of 38.7%, despite the fact that CIRT is a radiation therapy alone and has a short-term irradiation of eight times8.Since 1994, QST Hospital  has been providing carbon ion radiotherapy (CIRT)18F]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography/computed tomography (PET/CT) in determining efficacy has been reported in many studies13. [S-methyl-11C]-L-methionine (MET), mainly transported by system L amino acid transporters15, has been clinically used as a tumor-seeking agent for PET imaging for several decades16. MET PET/CT has fewer false positives due to inflammation and necrosis than FDG PET/CT17. MET PET/CT may be more helpful than FDG PET/CT or other imaging modalities in determining the efficacy of radiotherapy because radiotherapy is more likely to be associated with necrosis and inflammation than chemotherapy. The usefulness of MET PET/CT in radiotherapy has been reported in the diagnosis of relapse in brain tumors18, and its efficacy in lung and head and neck cancers has been assessed23. However, the clinical effectiveness of MET PET/CT in esophageal cancer has not yet been evaluated.For neoadjuvant therapy of esophageal cancer, the usefulness of 2-. As described shortly, CIRT is a radiation therapy alone without concurrent chemotherapy. The total dose ranged from 28.8 to 35.2 GyE, and single doses of 3.6 to 4.4 GyE were administered four times a week for eight times. External beam radiotherapy was delivered by two opposing anteroposterior and posteroanterior fields. The planning target volume was determined on CT scanning which included the whole primary tumor with a 3 cm margin in the craniocaudal direction and metastatic LNs with a minimum of a 1 cm margin. Consequently, all patients underwent radical esophagectomy at 4-week intervals after the completion of CIRT.25. Furthermore, the residual rate of cancer cells was quantified visually.Surgery was performed in all patients within 1 week of the last PET scan in Department of Frontier Surgery, Graduate School of Medicine, Chiba University. None of the patients showed local tumor progression or distant metastases during preoperative therapy. Surgical therapy consisted of transthoracic esophagectomy with three-field lymphadenectomy. Hematoxylin and eosin staining was performed for pathological evaluation, and all specimens were evaluated by two experienced pathologists who were blinded of the clinical and PET data. Tumor regressions were classified histologically based on the 11th edition of the Japanese Classification of Esophageal Cancer. Patients were divided into the following groups: Grade 1 (the disappearance rate of less than two-thirds in cancer cells), Grade 2 (the disappearance rate of more than two-thirds in cancer cells), and Grade 3 (the complete disappearance of cancer cells)Thirty-eight MET PET/CT scans were performed in all patients before and 3 weeks after the completion of CIRT. Twenty min after intravenous injection of 740 MBq (20 mCi) of MET, consecutively unenhanced CT scan was performed, followed by the emission images covering the area from the neck to the upper abdomen  were acquired for 30 min using a Biograph . The CT data were used for attenuation correction. The PET data sets were reconstructed using a filtered back-projection algorithm. Coregistered scans were displayed using the software .Volumes of interest (VOIs) of the tumor were outlined on the co-registered CT image of each patient when the registration images of the target lesion were matched between PET and CT images. The maximum and mean standardized uptake values (SUVmax and SUVmean), which are convenient semiquantitative methods and most widely used, were assessed for MET uptake by the lesion. Both SUVmax and SUVmean were measured pre- and post-CIRT. MET uptake consists of four parameters . The effect of neoadjuvant CIRT was investigated by examining the relationship between each parameter of MET uptake and histological efficacy .p values of less than 0.05 considered statistically significant. MET uptakes of Grades 1, 2, and 3 were compared by performing the nonparametric Tukey‒Kramer honestly significant difference test. MET uptakes of Grades 1, 2, and 3 were compared by performing the Mann–Whitney U test. The cutoff values were determined using the receiver operating characteristic curve. The best cutoff points for balancing the sensitivity and specificity of a test are the point on the curve closest to the  point. Optimal sensitivity and specificity are defined as those yielding the minimal value for (1 − sensitivity)2 + (1 − specificity)226. Linear regression between residual tumor rate and SUV was calculated, and Spearman’s correlation between the two groups was analyzed.All values were calculated as means ± standard deviations. Statistical analyses were performed using SAS JMP statistical software version 15 , with All procedures and subsequent analyses were performed with the approval of the Institutional Review Boards of Quantum and Radiological Science and Technology (QST) (reference number: 21–003). All enrolled patients received explanations, and then they provided written informed consent regarding this clinical study. Our study was conducted in accordance with the principles outlined in the 1964 Declaration of Helsinki and its later amendments.All 19 patients were diagnosed with squamous cell carcinoma, and the depths of tumor invasion were T1, T2, and T3 in 7, 7, and 5 patients, respectively . The pre-SUVmean were 4.33 ± 0.86, 5.46 ± 1.09, and 5.33 ± 1.17 for T1, T2, and T3, respectively .When the lesion uptake of MET was compared with depth of tumor invasion, pre-SUVmax were 6.90 ± 2.13, 9.41 ± 1.88, and 8.67 ± 2.36 for T1, T2, and T3, respectively (p < 0.005 for the difference between pre-SUVmax and post-SUVmax). Meanwhile, SUVmean were 5.01 ± 1.11 and 3.94 ± 0.80 for pre-SUVmean and post-SUVmean, respectively (p < 0.005 for pre-SUVmean and post-SUVmean).A summary of each case is described in Table n.s. for the difference between Grades 1 and 2, n.s. for Grades 1 and 3, n.s. for Grades 2 and 3; Fig. n.s. for Grades 1 and 2, n.s. for Grades 1 and 3, n.s. for Grades 2 and 3; Fig. n.s. for Grades 1 and 2, p < 0.05 for Grades 1 and 3, n.s. for Grades 2 and 3; Fig. p < 0.05 for Grades 1 and 2, p < 0.05 for Grades 1 and 3, n.s. for Grades 2 and 3; Fig. The relationship between MET uptake and all grades is shown in Fig. p = 0.87, difference between Grades 1, 2, and 3; Fig. p = 0.68, Grades 1, 2, and 3, respectively; Fig. p = 0.07 for Grades 1, 2 and 3; Fig. p = 0.08; Grades 1, 2, and 3; Fig. The relationship between MET uptake and Grades 1, 2, and 3 are shown in Fig. The accuracy of the differential diagnosis between the uptake of Grades 1, 2, and 3 is summarized in Table R2 = 0.01, correlation coefficient = 0.10, n.s.; Fig. X, R 2 = 0.08, correlation coefficient = 0.29, n.s.; Fig. X, R2 = 0.38, correlation coefficient = 0.62, p < 0.005; Fig. X, R2 = 0.32, correlation coefficient = 0.56, p < 0.05; Fig. The correlation between the residual tumor rate and uptake of MET is shown in Fig. Representative cases of MET PET/CT images for Grade 1 cases: T3N1M0, stage III and Grade 3 cases: T1bN0M0, stage I are shown in Fig. This study investigated the uptake of MET in esophageal cancer and its early response in neoadjuvant CIRT of esophageal cancer and its relationship with MET uptake on MET PET/CT.First, MET PET/CT imaging of esophageal cancer was clearly demonstrated. In terms of depth of tumor invasion, there was a tendency for the accumulation to be higher in T2 and T3 than in T1.12. In some cases, low uptake of MET was observed on the post-CIRT images, which were proved by the endoscopic examination after CIRT. Although MET PET/CT is considered to have fewer false positives due to inflammation than FDG PET/CT, there is also some degree of MET uptake into the inflammatory site17.In CIRT of esophageal cancer, MET uptake clearly decreased in response to CIRT. These findings are the first report of MET PET/CT in esophageal cancer. MET PET/CT was as useful as the reported regarding FDG PET/CT29. In radical chemoradiotherapy for stage II–III esophageal cancer, the CR rate was 70.6%, and 84% of them were non-recurrences29. Patients with CR after chemoradiotherapy are expected to have a relatively good prognosis without surgery.Chemoradiotherapy is as important as surgery in the treatment of esophageal cancer30. The advantage of avoiding surgery after radiotherapy is of paramount importance.In CIRT, there is a possible treatment option to avoid surgery in patients with CR. Therefore, it is essential to preoperatively diagnose Grade 3 cases after CIRT. In addition, neoadjuvant chemoradiotherapy had significantly higher rates of morbidity and mortality after surgery than neoadjuvant chemotherapyThe timing of MET PET/CT to diagnose histological efficacy was considered to be more useful approximately 3 weeks after CIRT, that is, just before or several weeks before surgery than before CIRT. Since the histological efficacy is determined from the surgical specimens after CIRT, it seems reasonable that MET uptake after CIRT close to the surgery best reflects the histological efficacy.The post-SUVmax was the most suitable parameter for the diagnosis of Grade 3, because the diagnostic accuracy of Grade 3 was the highest for post-SUVmax at approximately 80%.In this study, we attempted to quantify the histological efficacy as the residual tumor rate and to examine the histological efficacy in detail. When the correlation between residual tumor rate and MET uptake was examined, a positive linear relationship was observed for post-SUVmax, followed by the parameter using post-SUVmean.Since the residual tumor rate represents the density of the tumor, SUVmean is a theoretically ideal parameter for reflecting tumor activity. However, in this study, the prediction of residual tumor rate using SUVmean was found to be inferior to that using SUVmax. The issue of measurement accuracy in the SUVmean method was considered to be the reason for this. For a more accurate quantitative evaluation using SUVmean, it is essential to accurately determine the margins of the lesion. However, the CT component of PET/CT is non-contrast imaging, making it difficult to accurately determine the margin of the tumor lesion. In addition, because the esophagus is a luminal organ, semiquantitative evaluation is affected by the uptake value of the air portion of the esophageal lumen. Since the method used in the current study was a manual setting of the VOIs, it is not reproducible. Considering the reasons described above, the evaluation method using SUVmax is considered to be more appropriate and practical than that using SUVmean.If the residual tumor rate can be diagnosed, various treatment options are possible. If residual tumor rates are minimal, additional chemotherapy or chemoradiation for Grade 3 can be selected. If the residual tumor rates are low, less invasive surgical treatment, such as omitting cervical lymph node dissection, can be considered. If residual tumor rates are high, preoperative treatment may not be sufficiently effective, but other preoperative treatments can be added or modified. When residual tumor rates are high, the efficacy of preoperative treatment may be uncertain, but other treatments can be added or modified to improve the treatment effect.As described above, in CIRT for esophageal cancer, it was possible to diagnose local effects, such as tumor disappearance and tumor persistence, in detail by devising the timing of measurement and analysis method of MET accumulation. MET may be a useful imaging marker for the selection of other therapeutic options.This study has some limitations. First, the number of patients analyzed was small due to the number of cases set in this clinical trial. Second, since there were only two Grade 1 cases, further study is needed for a detailed comparison with histological effects. Third, prognostic analysis was not performed. In this clinical trial study protocol with limited number of patients and longer observation period, we are planning to determine clear indication criteria of CIRT for esophageal cancer by a combination of MET PET with other important prognostic factors.This is the first study to report the relationship between esophageal cancer and MET accumulation, and according to this study, MET PET/CT is feasible for the imaging of esophageal cancer. Furthermore, MET PET/CT is clinically useful for the assessment of early response to neoadjuvant CIRT in esophageal cancer. In particular, post-SUVmax in MET PET/CT is considered a promising PET imaging parameter.


To the Editor—Pharyngitis is one of the most common conditions leading to inappropriate antibiotic prescriptions. Rapid group A streptococcal (GAS) testing remains a key component of care to guide appropriate antibiotic prescribing for patients with acute pharyngitis. The percentage of pharyngitis encounters prescribed an antibiotic and that underwent GAS testing is also a key measure of the Healthcare Effectiveness Data and Information Set. In the initial months of the coronavirus disease 2019 (COVID-19) pandemic when personal protective equipment (PPE) was constrained, Intermountain Healthcare recommended limiting rapid GAS testing in urgent care clinics to preserve PPE. We describe our experience with pharyngitis encounters and the impact of temporarily reducing GAS testing on antibiotic prescribing for pharyngitis before and during the COVID-19 pandemic.International Classification of Diseases, Tenth Edition, Clinical Modification (ICD-10) codes  and a validated methodology. Encounters were assessed for antibiotic prescriptions ordered through the electronic health record (EHR) and the use of point-of-care rapid GAS tests. Pharyngitis encounters were analyzed monthly by assessing the percentage of encounters associated with an antibiotic prescription regardless of testing and the percentage of encounters associated with an antibiotic prescription when a GAS test was or was not performed. Three periods relating to COVID-19 and GAS testing recommendations were examined: prepandemic (July 2018–March 2020), pandemic onset (April 2020–June 2020), and the pandemic (July 2020–August 2021).We performed a retrospective cohort study of pharyngitis encounters within the Intermountain Healthcare urgent-care network from July 2018 through August 2021. Intermountain Healthcare is a nonprofit, vertically integrated healthcare delivery system with 38 urgent-care clinics in Utah. Moreover, 32 of these clinics provide care for patients of all ages and 6 provide care exclusively to children aged <18 years. All provide only in-person care and do not provide telehealth services. On April 1, 2020, urgent-care leadership recommended limiting rapid GAS testing to children aged ≤15 years and empirically treating most adults where GAS was considered likely. More specific guidance was not offered. In late May 2020, urgent-care leadership instructed clinicians to resume routine testing practices, which strongly encourage testing when prescribing antibiotics. We identified all urgent-care encounters associated with a primary diagnosis of pharyngitis using 1 and 2.In total, 115,558 encounters for pharyngitis were identified. Most patients were white (92.7%) and 59.2% were female. The average age was 25.0 years: 74,107 (64.1%) were aged ≥18 years and 38,776 (33.6%) were aged 3–18 years. During the prepandemic period, the monthly percentage of pharyngitis encounters for which rapid GAS testing was performed, regardless of an antibiotic prescription, was consistently near 90%. The average percentage of monthly pharyngitis encounters prescribed an antibiotic that also underwent GAS testing was 90.4%. Following the recommendation to limit testing, the monthly percentage of pharyngitis encounters for which rapid GAS testing was performed sharply declined from 84.2% in March 2020 to 37.9% in April 2020. The average monthly percentage of pharyngitis encounters in urgent care in which an antibiotic was prescribed increased from 38.9% to 50.6% during this pandemic onset period. In the prepandemic period, an average of 9.6% of pharyngitis encounters were prescribed an antibiotic without GAS testing, and this rose to 70.2% during the pandemic onset period. The monthly percentage of pharyngitis encounters in urgent care for which rapid GAS testing was performed returned to levels ≥80% by July 2020 as routine testing practices were restored. The average percentage of monthly pharyngitis encounters prescribed an antibiotic that also underwent GAS testing rose to 87.3% during the pandemic period. Additional patient demographics and testing characteristics are available in Supplementary Tables As rapid GAS testing volumes decreased in the urgent-care centers of our healthcare network due to limited PPE during the initial months of the COVID-19 pandemic, we observed a simultaneous relative increase of >30% in antibiotic prescribing for pharyngitis driven by an increase in antibiotic treatment for pharyngitis without testing . An abrupt rise in GAS infections is also unlikely because the incidence of other respiratory infections in general declined with social-distancing measures. The observed decrease in antibiotic prescribing for pharyngitis encounters to levels below those preceding the pandemic onset may be explained in part by the simultaneous introduction of an urgent-care–based antibiotic stewardship program intervention between July 2019 and June 2020.


Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA double–strand breaks (DSBs), to yield long 3′ ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single–molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N–terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis–dependent strand annealing (SSA) instead of HR–mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination. Bloom syndrome is a genetic disorder associated with increased cancer risk and is caused by mutations in Bloom helicase. This study investigates the mechanisms used by BLM helicase as initiates the repair of broken chromosomes. Aberrant HR underlies the chromosomal rearrangements often associated with cancers, cancer prone syndromes, and numerous genetic diseases5. During the early stages of HR, DSB ends are processed by 5′→3′ strand resection, yielding 3′ single-stranded DNA (ssDNA) overhangs that become coated with the heterotrimeric ssDNA-binding protein replication protein A (RPA). RPA is then replaced by the ATP-dependent recombinase RAD51 to form a nucleoprotein filament, termed the presynaptic complex, capable of catalyzing DNA strand invasion during which the RAD51-bound ssDNA is paired with a homologous double-stranded DNA (dsDNA) donor template5. DNA synthesis then occurs within the resulting D-loop structure, and repair is completed via one of several mechanistically distinct pathways5.Homologous recombination (HR) is an important pathway for repairing DNA double-strand breaks (DSBs), single-strand gaps, and stalled or collapsed replication forks8. In humans, long-range DNA end resection is catalyzed by the Bloom helicase  in combination with either DNA2 or EXO110. Germline mutations in BLM are associated with Bloom syndrome, an autosomal recessive genetic disorder characterized by severe developmental defects and strong cancer predisposition13. Moreover, BLM and RAD51 are often overexpressed in many cancer types14. In addition to its DNA end processing role, BLM has been implicated in the disruption of inactivated ADP-bound RAD51 filaments and strand invasion intermediates17, Topoisomerase IIIα–dependent dissolution of the double Holliday junction (dHJ) that arises during some HR events18, and in the restart of stalled or collapsed replication forks23. Given its importance to the maintenance of genome integrity, BLM has emerged as a potential target for anticancer chemotherapeutics25.In addition to creating a ssDNA template for RAD51 presynaptic complex assembly, extensive DNA end resection also plays a crucial role in committing break repair via HR rather than nonhomologous end joining28. BLM has DNA-dependent ATPase and 3′→5′ helicase activities and is capable of unwinding DNA structures mimicking a variety of DNA replication and repair intermediates29. In addition to its helicase core domain, BLM harbors a RecQ C-terminal (RQC) domain that confers high-affinity structure-specific DNA-binding activity, and a helicase- and RNaseD-like C-terminal (HRDC) domain that promotes BLM recruitment to DNA damage31. In vitro studies have provided insights into BLM activities relevant for its involvement in DNA end resection6 and dHJ dissolution18. However, our current understanding of BLM function(s) remains relatively limited at the mechanistic level. Given that BLM is indispensable for multiple aspects of genome integrity, it remains challenging to fully define how BLM mutations impact any one particular process.BLM (1417 amino acid residues) belongs to the highly conserved RecQ subgroup of the super-family 2 helicasesHere, we used total internal reflection fluorescence microscopy (TIRFM) to define the interactions of N-terminal domain (NTD) and C-terminal domain (CTD) deletion BLM mutants with DNA. Our results confirm that the NTD is involved in BLM oligomerization and also show that the NTD allows BLM to generate a large ssDNA loop while unwinding duplex DNA. Surprisingly, while an NTD truncation mutant loses the ability to oligomerize, it is more proficient than full-length BLM at DNA end resection in vitro. However, cells expressing this NTD truncation mutant are sensitive to DNA damaging agents, and although these cells undergo DNA end processing, they are defective for DNA damage-induced RAD51 focus formation and the processed DSBs are channeled into the synthesis-dependent strand annealing (SSA) repair pathway instead of HR-mediated repair. We also demonstrate that there is a region within the CTD that is necessary for targeting BLM to DNA ends. Even though deleting amino acid residues of BLM needed for DNA end recognition has little or no impact on translocase and helicase activities, the CTD mutant fails to selectively bind DNA ends, which leads to a defect in end resection activity both in vitro and in vivo. Together, our study provides insights regarding the multi-faceted role of the BLM helicase in DSB repair by HR.35. DNA molecules were anchored via a biotin-streptavidin linkage to a lipid bilayer deposited onto a flow-cell surface and aligned at nanofabricated chromium (Cr) barriers using buffer flow, as previously described  co-localized with the free end of DNA molecules  BLM monomers bound per DNA end  molecules with a 30-nucleotide (nt) ssDNA 3′ overhang as a substrate, which mimics a partially processed DSB based upon the preference of BLM for 3′ ssDNA overhangs during dsDNA unwindingbed Fig. 36. BLM wles Fig. . Photobl39. When DNA2 was injected into the flow cell, the end-bound GFP-BLM translocated away from DNA ends at rate of 10 ± 8 bp s–1 (N = 48) and traveled an average distance of 7.1 ± 2.7 kb (N = 48) before stopping  and 10.8 ± 5.0 kb (N = 76) in reactions that included both DNA2 and RPA  of observed end-bound BLM molecules underwent translocation within the observation time windows. However, 20.3% (N = 48/237) and 25.5% (N = 64/251) of observed BLM translocated on the DNA when either DNA2 or RPA were present, respectively. In reactions containing both DNA2 and RPA, 36.9% (N = 76/206) of the end-bound BLM underwent translocation. The translocation velocity and processivity of GFP-BLM, and the stimulatory effects of DNA2 and RPA on these parameters, were comparable to previous reported values40.As we and others have previously reported, GFP-BLM readily undergoes translocation when bound to an internal position of a dsDNA molecule–1 with YOYO-1 vs. 10 ± 8 bp s–1 without YOYO-1; N = 35; p = 0.20), however, the processivity of end resection was lower when YOYO-1 was present  . However, some of the end-bound RPA-mCherry puncta underwent extension abruptly in a single-step event  molecules were labeled with ATTO565, allowing us to track the fate of the DNA ends Fig. .Fig. 2FaN = 102 of 143 for 3′-end; 80.0%, N = 107 of 134 for 5′-end) co-localized with the free DNA ends for both 3′ and 5′ ATTO565 end-labeled substrates, indicating that the dye does not affect end recognition by BLM  quickly disappeared , the 5′ end-labeled ATTO565 traveled together with GFP-BLM, indicating that it was not cleaved by DNA2  and processivity of 10.1 ± 5.5 kb  remained closely co-localized with GFP-BLM for the 30-min duration of our observations, with the remainder showing evidence of 3′ end release  compared to reactions that contained GFP-BLM, RPA-mCherry and DNA2 , suggesting that DNA2 may also contribute to continued retention of the 3′ ssDNA end during resection.Next, we sought to determine if BLM alone was sufficient for ssDNA loop formation in the absence of DNA2. For these experiments, we injected RPA-mCherry into a sample chamber containing end-bound GFP-BLM in buffer with 2 mM ATP. As expected, the RPA-mCherry signal increased with time indicating that BLM translocation coincided with the continuous generation of ssDNA. Importantly, the RPA-mCherry signal remained in highly condensed puncta that co-localized with GFP-BLM Fig. , and anaase Fig. . NotablyN = 69; Fig. N = 67/69), the 3′ end remained co-localized with GFP-BLM throughout the reaction , the 3′ end was released suddenly from GFP-BLM , the 5′ end of the DNA did not co-localize with GFP-BLM as reflected by a clear spatial separation between the ATTO565 and GFP signals  yielded a truncated protein that was still efficiently targeted to the free DNA ends  and similar results were obtained for the other six NTD deletion proteins  and 131% further  than full-length BLM in reactions without DNA2  than that of full-length BLM  of the end-bound GFP-BLMΔ1–641 molecules exhibited single-step photobleaching behavior, suggesting that just one molecule of GFP-BLMΔ1–641 was bound , although two mutants (BLMΔ236–333 and BLMΔ334–431) showed ≥54% reduction in signal intensity (p < 0.0001) relative to full-length BLM, suggesting that multiple contacts within the NTD contribute to BLM oligomerization at DNA ends 17. This latter finding is consistent with prior reports indicating that deletion of the HRDC partially decoupled ATP hydrolysis from duplex DNA unwinding, resulting in nonproductive ATP hydrolysis cycles47.We next tested a series of C-terminal truncation mutants to assess their potential impact upon DNA end processing Fig. . The truues Fig. . InteresDNA Fig. . Despite∆1290–1417, which lacks the BLM C-terminal region but retains the HRDC domain  BLM∆1290–1330 which exhibited a complete loss of end-binding specificity but retained DNA translocase activity in the DNA curtain assays.We next sought to further characterize the properties of some of the BLM mutants in bulk biochemical assays, focusing efforts on: (1) BLM9. The NTD truncation mutant BLM∆1–641 had a 72% increase in dsDNA unwinding ability compared to full-length BLM, consistent with its increased velocity and processivity in the single-molecule assays  relative to full-length BLM  followed by siRNA knockdown of endogenous BLM and EXO1 expression  compared to cells expressing full-length GFP-BLM . 53BP1 is an early marker of DSBs, and comparable levels of IR-induced 53BP1 foci were observed among cell types that express the various BLM species Fig. . GFP-BLMect Fig. . These dion Fig. . In sharoci Fig. . Taken t49. For these assays, U2OS cells depleted for endogenous BLM and EXO1 expression by siRNA knockdown and harboring integrated copies of either the DR-GFP or the SA-GFP cassette were transformed with BLM expression constructs  and a plasmid encoding I-SceI ceI Fig. . ControlceI Fig.  and alsoceI Fig. . NotablyBLM Fig. . In control Fig. . Moreovells Fig. . To furtHere, we have examined the impact of NTD and CTD truncation mutations on the biochemical attributes of BLM and on DNA end resection in conjunction with DNA2 and RPA. Our data suggest that DNA end resection coincides with the formation of a large ssDNA loop and suggests that productive end resection may involve multimeric forms of the BLM helicase. Our findings have important implications for understanding the mechanisms of DNA end resection in metazoans.S. cerevisiae Sgs1, which binds DNA ends in an inactive apo configuration that can be activated by DNA2 or RPA35. Interestingly, even with both DNA2 and RPA present, end-bound BLM still translocated much more slowly compared to BLM initially bound to an internal region of dsDNA 40. In addition, we also find that the presence of DNA2 can greatly slow the translocation velocity of end-bound BLM (~10–15 bp s–1 vs. ~40 bp s–1), as has been previously observed40. These results suggest that the translocation characteristics of BLM are drastically different during DNA end processing compared to reactions in which BLM is simply unwinding a dsDNA molecule from an internal location, consistent with the premise that the structure of the bound DNA, together with the presence of DNA2, have important regulatory impacts upon BLM.Our data help to define the molecular mechanisms underlying BLM–DNA2-dependent DNA end resection and the role of BLM in downstream steps of HR. Our results suggest that initial DNA end recognition by BLM results in the formation of a stable end-bound complex that becomes activated for end resection upon addition of DNA2, RPA or both. The involvement of DNA2 and RPA in activating end-bound BLM for translocation is very similar to what we have previously reported for 23, as the resection machinery progresses along the dsDNA. Such a mechanism for ssDNA loop formation by human BLM bears some similarity to the DNA end-binding activity of Rep, NS3, PcrA, Srs2, Pif1 and several other helicases, which enables the helicase to undergo repetitive shuttling behavior on DNA fragments as revealed in smFRET studies54.We also find that end resection coincides with the formation of a large ssDNA loop or loop-like structure that is coated with RPA. BLM itself is primarily responsible for DNA loop formation, presumably by maintaining contact with the 3′ ssDNA end, or perhaps by maintaining contact with RPA bound at or near the 3′ ssDNA end (see below)∆1–641, which is proficient for DNA end resection in vitro and in vivo but does not support formation of RAD51 foci in cells exposed to IR. These findings also imply that BLM itself may play a more direct role in loading RAD51 onto newly resected ssDNA than previously appreciated and suggests the possibility that end resection and presynaptic complex formation may be temporally coordinated through the actions of BLM.One can envision a number of possible benefits of coupling DNA end resection to ssDNA loop formation. For instance, formation of an ssDNA loop may help to protect the 3′ ssDNA end to ensure that it is not degraded by other cellular nucleases. Formation of a ssDNA loop may also help to coordinate other downstream events such as the recruitment of protein components during formation of the presynaptic complex, including proteins such as BRCA1–BARD1, BRCA2, RAD51 and RAD52. A model in which end resection is somehow linked to presynaptic complex assembly is supported by experiments with the mutant BLMOur findings demonstrate the NTD encompassing amino acid residues 1–641 exerts a clear impact upon BLM oligomerization and ssDNA loop formation during DNA end resection. It seems possible that BLM oligomers possess a multiplicity of DNA-binding domains to allow for stable interactions with the newly generated ssDNA even as the end resection machinery travels along the DNA substrate. For example, if one BLM protomer drives ATP-dependent translocation, then one or more of the other protomers within the oligomeric complex may remain stably bound to the end of the unwound ssDNA without undergoing translocation.∆1–641 translocates on DNA at a faster rate on dsDNA and with higher processivity than full-length BLM, suggesting that assembly of higher order BLM oligomers affects the helicase activity of BLM negatively. Moreover, our data show that BLM∆1–641 supports end resection in vivo as evidenced by the appearance of RPA foci. In sharp contrast, BLM∆1–641 fails to support RAD51 focus formation in vivo and I-SceI induced DSBs are channeled through SSA-mediated DNA repair instead of HR, consistent with reduced RAD51 filament formation in cells expressing BLM∆1–641. As we have speculated above, the NTD of BLM may be somehow involved in loading RAD51 onto processed DNA breaks. In vitro DNA resection assays using BLM and EXO1 have also led to the suggestion that RAD51 loading onto the resected DNA ends may be facilitated through protein–protein interactions between RAD51 and BLM55. Notably, BLM and RAD51 co-immunoprecipitates from cellular extracts and a combination of two-hybrid and Far Western analyses mapped RAD51 interaction regions to the N-terminus (amino acid residues 1–212) and C-terminus (amino acid residues 1317–1417) of BLM56. The mutant BLMΔ134–235 supports DNA end looping  is involved in BLM dimerization and possibly hexamer formation44. Surprisingly, we have found that when amino acid residues spanning this region are deleted (i.e. BLMΔ334–431), we could still see some evidence for BLM oligomerization at DNA ends, albeit to a significantly lesser extent than is observed for full-length BLM. Indeed, all of our BLM constructs, with the exception of BLMΔ1–641, show evidence for some oligomerization at the DNA ends. Interestingly, by electron microscopy, BLM has been shown to exhibit a marked tendency to oligomerize through protein–protein interactions on DNA substrates mimicking D-loops with a 5′ invading end59. Comparable measurements have not been made on DNA substrates mimicking end processing intermediates. Thus, our data may reflect some propensity of multiple BLM monomers to interact with one another while bound to DNA ends.BLM can oligomerize in solution but dissociates into monomers upon addition of ATP62. Of the five human RECQ helicases, BLM and WRN possess this domain, whereas RECQ1, RECQ4 and RECQ5 do not62. The HRDC is necessary for targeting BLM to sites of DNA damage in vivo31, but there are conflicting reports regarding its involvement in vitro in DNA-binding64. The crystal structure of BLM (residues 640–1298) bound to DNA shows that the HRDC domain is ~28 Å away from the DNA47. Thus, one interpretation of the cellular data is that HRDC helps target BLM to DNA damage not through direct contacts with DNA, but instead through protein–protein contacts with some other DNA repair factor. We find that deletion of the entire BLM CTD from amino acids 1207 to 1417 (i.e. BLM∆1207–1417), which includes the HRDC, abolishes DNA end recognition in vitro, but this mutant protein is still able to bind internal positions along the dsDNA. Interestingly, even though BLM∆1207–1417 exhibits a marked increase in ATPase activity, its translocase attribute is significantly impaired (54% lower) compared to full-length BLM. Together, these results suggest that removal of the HDRC domain partially decouples BLM ATP hydrolysis from DNA translocation, resulting in futile cycles of ATP hydrolysis that do not result in protein movement along the DNA, as has been suggested previously47.The function of the HRDC domain located near the BLM CTD has been somewhat enigmatic∆1291–1330) abolishes DNA end recognition in vitro, but unlike the removal of the entire HRDC domain, deleting this short peptide has little or no impact on BLM ATPase or translocase activity. Remarkably, despite its inability to target DNA ends in vitro, BLM∆1291–1330 does form IR-dependent foci in cells, suggesting that it is recruited to DNA damage. However, RPA and RAD51 focus formation was severely compromised in cells expressing BLM∆1291–1330, indicating that this mutant was unable to support normal DNA end resection in vivo. Thus, while BLM∆1291–1330 can be recruited to DNA damage through specific protein–protein interactions with other repair factors, perhaps involving the HRDC domain31, it is unable to productively initiate DNA processing due to its DNA end recognition defect. We favor a model in which amino acid residues 1290 to 1330, perhaps together with the flanking HRDC domain65, may exert an allosteric or structural influence over the RQC domain, the helicase core domain, or both domains, which modulates their ability to specifically recognize DNA ends.We have also identified a conserved peptide sequence encompassing amino acid residues 1290 to 1330 that is essential for BLM recruitment to DNA ends. This peptide sequence flanks the HRDC domain but is not a part of the HRDC domain itself. Deletion of these amino acid residues , although we note that this model for ssDNA loop formation is distinct from our present results with the human enzymes, because we show that DNA2 is not necessary for ssDNA loop formation and the 3′ end of the ssDNA remains associated with the resection machinery as it travels along the dsDNA.We have previously reported our DNA end resection studies using the E. coli RecBCD and B. subtilis AddAB helicase/nuclease complexes, although the overall mechanisms of the human and these bacterial end resection machineries are quite distinct72. As with the BLM–DNA2 system, it is not yet clear whether ssDNA loop during end resection in bacteria is an essential part of DNA repair in bacteria. However, protein–protein interactions between the RecB subunit of RecBCD and RecA facilitate RecA loading onto the ssDNA strand generated during end resection77, although it is as yet unknown whether RecA loading might be influenced by or coupled to ssDNA loop formation. Given the above, it will be of great interest to examine the potential relationship between ssDNA loop formation during DNA end resection and presynaptic complex formation mediated by BLM–DNA2, RecBCD and AddAB.Interestingly, our model for end resection by BLM–DNA2 bears some resemblance to what takes place during DNA end resection by the 81. Moreover, within the cellular setting, DNA end processing is likely to be intimately coupled to assembly of the RAD51–ssDNA presynaptic complex, and our data suggest that BLM itself may help couple end processing and RAD51 loading. The assays presented here offer a valuable tool to define the impact of accessory factors on DNA end processing and presynaptic complex assembly.Our data reveal mechanistic insights into BLM as it fulfills its role in DNA end processing in conjunction with DNA2 and RPA. We note that the activity of BLM in DNA end resection is also influenced by other proteins, including the TOP3–RMI complex, the MRE11–RAD50–NBS1 complex, and CtIP17. For BLM transient transfection in siBLM/siEXO1 U2OS cells, the BLM constructs were subcloned into the pBI–EGFP vector using Gibson assembly.BLM truncation mutants were generated through divergent PCR cloning using GFP-BLM–pYES2 as a template83. GFP–BLM truncation mutants were purified similarly to full-length GFP–BLM, with some minor modifications. In brief, the GFP–BLM mutant expression plasmids were transformed into a protease-deficient yeast expression strain (JEL–1). Cells were grown in six liters of basic medium minus uracil  to 1.0 OD600 at 30 °C and then induced overnight at 25 °C with the addition of 2% galactose. Cells were harvested by centrifugation and lysed by vortexing in 40 ml cell lysis buffer , 2 mM EDTA) with the same volume of glass beads  at 4 °C. All cell lysates were precipitated with 20% ammonium sulfate (10 g per 50 ml supernatant). After 1 h, the protein pellets were recovered by centrifugation at 13,000 × g at 4 °C for 10 min. The protein pellets were then dissolved in 20 ml cell lysis buffer without EDTA plus 25 mM imidazole. These protein resuspensions were passed through a 0.45 µm filter  to remove any undissolved precipitate. The protein solutions were then purified using Ni-NTA (Qiagen) resin and eluted with an imidazole step gradient using successive washes of buffer containing 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200 and 250 mM imidazole and peak fractions containing GFP–BLM were identified by SDS-PAGE and Coomassie staining. The purified GFP–BLM fractions were combined aliquoted and stored at –80 °C.RPA, RPA-mCherry, RPA-GFP, DNA2, GFP-BLM, and GFP-BLM (K695A) were purified as previously described17. For dsDNA with a 30-nt 3′-end tail, λ-DNA  was annealed to a biotinylated oligo (5′-Phos-AGG TCG CCG CCC-3′BioTEG), and the second end of the λ–DNA was annealed to the oligonucleotide with the following sequence: 5′-Phos-GGG CGG CGA CCT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-3′ to yield a dsDNA substrate with a 3′ 30-nt poly(dT) overhang, as previously described35. For dsDNA with the 30-nt 3′ end tail end-labeled with ATTO565, the λ-DNA was annealed the biotinylated oligo as described above, and the second end of the λ–DNA was annealed to the oligonucleotide with the following sequence: 5′-pGGG CGG CGA CCT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-ATTO565N-3′. For dsDNA with a 30-nt 5′ end tail labeled with ATTO565, λ–DNA was annealed to the biotinylated oligo, and the second end of the λ–DNA was annealed to two oligonucleotides with the following sequence: 5′-ATTO565N-AGT GTC GTG CCG G-3′ and 5′-Phos-GGG CGG CGA CCT CCG GCA CGA CAC TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T-3′. All substrates were ligated overnight using T4 DNA ligase  and purified by PEG precipitation, as described17.The λ-DNA substrates were prepared as previously describedThe 2.5 kb dsDNA substrate used for the bulk biochemical assays was a PCR amplified fragment of pFastBac–HTB generated using PCR primers with the following sequence: 5′-ATTO532N-ATC ACT GAT ATC GCC TAG G-3′ and 5′-ATTO532N-ACC AAT GCT TAA TCA GTG AGG-3′. After the PCR reaction, the substrates were purified by electrophoresis in a 0.8% agarose gel stained with SYBR Safe and purified using Wizard SV gel clean-up system 2, 2 mM ATP, 1 mM DTT, 0.2 mg/ml BSA, 10 mM creatine phosphate, 0.05 mg/ml creatine kinase, and 200 nM RPA) with 1.5 nM DNA ends (2.5 kb dsDNA substrate) at 37 °C for 15 min. Reactions were initiated by adding 2.5, 5, or 10 nM BLM with or without 30 nM DNA2 and stopped by adding SDS to 0.2% and protease K to 0.25 mg/mL. Then reaction mixtures were incubated for another 5 min at 37 °C. The resulting samples were loaded onto a 1% agarose gel 1xTAE buffer. Gels were scanned using a Typhoon imager  and the intensity of the dsDNA and ssDNA bands was quantified using ImageJ.BLM helicase and BLM–DNA2 nuclease assays were performed in BLM helicase buffer  at 37 °C with M13 ssDNA . Reactions were initiated by the addition of 5 nM BLM variants, as indicated. Aliquots were removed at the indicated time points and quenched by addition of 50 mM EDTA. The quenched reactions were quantified by the ATPase/GTPase Activity Assay Kit as per the manufacturer’s instructions .ATPase assays for BLM variants were performed in BLM buffer , 0.5% biotinylated-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine , and 8% mPEG 2000-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4- cyclohexane-carboxamide]  and deposited onto the surface of flowcell sample chamber, as described84. The biotinylated λ–DNA substrates were then injected into the sample chamber and attached to the bilayer through a biotin-streptavidin linkage. The flowcell was then connected to a microfluidic system and sample delivery was controlled using a syringe pump (Kd Scientific)84. Biotinylated dsDNA substrates were aligned at the barriers by application of flow in BLM buffer , as indicated) at 37 °C with a flow rate of 0.15 ml/min.All experiments were conducted with a prism-type total internal reflection fluorescence microscope (Nikon) equipped with a 488-nm laser , a 561-nm laser , and two Andor iXon EMCCD cameras84. Data acquisition was controlled using NIS-Elements Version 5.1 (Nikon). Image acquisition was started immediately before the protein injections and the illumination lasers were shuttered between each acquired image to minimize photobleaching. GFP–BLM or the GFP–BLM truncation mutants (0.2 nM) in BLM buffer were injected into the flow cell through a 150 µl loop. Then, 2 ml BLM washing buffer  was injected into the sample chamber with a flow rate of 1 ml/min. Reactions were initiated by injection of the indicated proteins  in BLM buffer at 0.25 ml/min. For experiments using both GFP–BLM and RPA-GFP, the GFP–BLM was photobleached prior to the injection of RPA-GFP.For experiments, images were collected at 1 frame per 20 or 30 s with 0.1 s integration time using two EMCCD cameras with a custom-built shuttering system to avoid signal bleed-through during image acquisitionhttp://imagej.net/Contributors). Raw TIFF images were imported as image stacks into ImageJ and images were corrected for drift using the StackReg function in ImageJ84. Kymographs were then generated from the corrected image stacks by defining a 1-pixel wide region of interest encompassing individual dsDNA molecules, and these kymographs were used for analysis of GFP–BLM processivity, velocity and binding distributions as previously described35.Single-molecule image analysis was performed using Open-source image processing software ImageJ  supplemented with 10% V/V Fetal Bovine Serum  and 1% V/V penicillin-streptomycin antibiotic .85 containing WT-BLM, BLMΔ1–641 and BLMΔ1291–1330 were used to transfect the U2OS cells in a 6 well using Lipofectamine 3000 Transfection Reagent . In total, 25 nM of siRNA mix containing siBLM , siEXO1  or the siScramble control Dharmacon ON-TARGET plus Non-Targeting Control siRNA 1 Cat. No.: D–001810–01–05) were added to the cells 24 h after plasmid transfection, using RNAiMax regent Lipofectamine™ RNAiMAX Transfection Reagent .pBI–MCS–EGFP 2, 1 mM DTT, 0.1% Triton X-100) and fixed with paraformaldehyde (PFA) for 15 min at RT. Next, cells were permeabilized for 15 min at RT and incubated with blocking buffer  for 1 h followed by addition of primary antibody against RAD51 , RPA , Abcam, Cat. No.: ab2175; used at a 1:500 dilution), or 53BP1  O/N at 4 °C. Cells were washed three times with phosphate-buffered saline (PBS) for 5 min each and stained with an Alexa Fluor 594 labeled secondary antibody  for 1 h at RT followed by 3 more PBS washes. Coverslips were mounted using ProLong Diamond Antifade Mountant with DAPI  and sealed. Images were acquired on an Olympus FLV3000 inverted laser scanning microscope using acquisition software FV31S-SW. For visualizing GFP–BLM foci, cells were exposed to 8 Gy IR and then fixed with a 2% PFA solution for 15 min at RT. PFA was replaced then with PBS. Slides were mounted and imaged using confocal microscope using ×60 oil immersion objective. For foci analysis, cells with more than 5 foci were considered positive and at least 300 cells were counted over three independent experiments. Image analysis was performed with Analysis FV31s-DT software. Data analysis and graphs were generated with GraphPad Prism version 8.4.3. p values were calculated using one-way ANOVA test using SEM.Cells were treated with 8 Gy radiation and collected after 4 h of incubation at 37 °C. Pre-extraction was done for 10 min on ice with CSK Buffer  and protein quantitation was done using the Pierce BCA Protein Assay Kit . In total, 50 µg protein samples were run on 4–15% SDS-PAGE gels, protein from the gels were transferred onto NC membrane . NC membranes were blocked using 5% dry milk in TBST  and stained with primary antibodies: anti-BLM , anti-EXO1, anti-FLAG (M2) , anti-Actin , anti-GFP , or Alpha–Tubulin–HRP , as indicated, O/N at 4 °C, washed with TBST buffer 3 times for 10 min each and incubated with appropriate secondary antibody  for 1 h at RT. Membrane was washed three times for 10 min each and developed using Pierce Enhanced Chemiluminescence substrate . Blots were imaged on a BioRad Chemidoc imaging system using Image Lab Touch software version 2.4.0.03.86. Briefly 150 Cells were seeded per well in a 12 well plate and treated with the indicated concentrations of Olaparib  and MMC  and allowed for colony formation for 10 days. Colonies were fixed with methanol:acetic acid (3:1) for 5 min and stained with 0.5% crystal violet in methanol for 15 min. Plates were washed under tap water and dried before colonies were counted. Cells were imaged on a BioRad Chemidoc imaging system using Image Lab Touch software version 2.4.0.03.Clonogenic survival assays were performed as described49. Briefly, U2OS cells harboring integrated copies of either DR-GFP or SA-GFP cassette were transfected with siRNA (siBLM + siEXO1) or Scr-siRNA overnight and complemented with vector control (pBI-MCS-Flag), WT-BLM, BLMΔ1–641 and BLMΔ1291–1330, along with plasmid expressing I-SceI endonuclease. Cells were incubated 72 h post plasmid transfection and % GFP positive cells were analyzed using flow cytometry (BD LSR-II). Repair efficiency was calculated as p values were calculated using one-way ANOVA test using SEMCell-based reporter assays were performed as previously described50. Briefly, ER-AsiSI U2OS cells were depleted of endogenous BLM and EXO1 using siRNA and then complemented with WT-BLM, BLMΔ1–641, or BLMΔ1291–1330. Cells were then treated with 300 nM 4-Hydroxytamoxifen  for 4 h to allow the nuclear translocation of AsiSI and the induction of DSBs. Cells were harvested and genomic DNA extracted using a DNeasy Kit (Qiagen). After that, 1500 ng of genomic DNA was digested, or mock digested, with 2 μl of BsrGI enzyme  in 30 μl total volume at 37 °C overnight. In total, 1 μl of DNA was used as template in 10 μl of qPCR reaction performed using SYBR Green master mix  and 0.5 μM of each primer . qPCR data were quantified on a 7900 HT Fast Real-Time PCR system (Applied Biosystems) using SDS2.4 software. For each sample, ∆p values were calculated using unpaired t test.The percentage of DNA resection adjacent to a specific DSB, DSB1 (Chr 1: 89231183) was measured as previously describedN) are specified in each binding distribution histogram. For the single-molecule BLM velocity and processivity data error bars represent standard deviation (SD) and the N values are presented in each corresponding figure panel. The statistical parameters  for all velocity and processivity measurements are summarized in Supplementary Table N values are presented in the figure panel and error bars represent SD from three independent experiments. The analysis shown in Supplementary Fig. N values for all single-molecule experiments represent the number of single molecules that were analyzed for each given experiment and reflect the cumulative data collected from at least three separate flowcells. p values for the single-molecule velocity and processivity assays (Figs. t test. For the bulk biochemical ATP hydrolysis (Fig. p values for the biochemical assays were calculated using a two-tailed Student’s t test.For the single-molecule BLM binding distribution analysis and error bars represent 95% confidence intervals calculated from bootstrap analysis of the data. The number of single BLM molecules measured (ys Figs.  were calsis Fig. , DNA unwsis Fig.  and DNA sis Fig.  the erroN equals three independent experiments. For each experiment, cells were scored as positive if they contained greater than five foci per cell and ≥300 cells were analyzed for each different condition. For reporter assays (Fig. p values for cellular assays were calculated using one-way ANOVA test using the SEM.For cell survival assays Fig. , the datays Fig.  the % GFp values were calculated using an unpaired Student’s t test using the SEM.For PCR-based resection assays (Supplementary Fig. Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary InformationReporting summary


In the United States, children aged <5 years receive high volumes of antibiotics, which may contribute to antibiotic resistance. It has been unclear what role preventable illnesses and chronic comorbidities play in prompting antibiotic prescriptions.We conducted an observational study with a cohort of 124 759 children aged <5 years born in the United States between 2008 and 2013 with private medical insurance. Study outcomes included the cumulative number of antibiotic courses dispensed per child by age 5 and the proportion of children for whom at least 1 antibiotic course was dispensed by age 5. We identified which chronic medical conditions predicted whether a child would be among the top 20% of antibiotic recipients.Children received a mean of 6.8  antibiotic courses by age 5, and 91% (95% CI: 90%–92%) of children had received at least 1 antibiotic course by age 5. Most antibiotic courses  were associated with respiratory infections. Presence of a pulmonary/respiratory, otologic, and/or immunological comorbidity substantially increase a child's odds of being in the top 20% of antibiotic recipients. Children with at least 1 of these conditions received a mean of 10.5 (95% CI: 10.4–10.6) antibiotic courses by age 5.Privately insured children in the United States receive many antibiotics early in life, largely due to respiratory infections. Antibiotic dispensing varies widely among children, with more antibiotics dispensed to children with pulmonary/respiratory, otologic, and/or immunological comorbidities. Privately insured children in the United States receive many antibiotics early in life, largely due to respiratory infections. Antibiotic dispensing varies widely among children, with more antibiotics dispensed to children with pulmonary/respiratory, otologic, and/or immunological comorbidities. The clinical effectiveness of antibiotics is increasingly threatened by antibiotic resistance, generated through evolutionary selective pressures exerted by antibiotic consumption . To dateTo establish a disease-prevention strategy aimed at reducing antibiotic consumption in a population, it is necessary to first quantify the baseline volume of antibiotic consumption, both overall and stratified by specific pathogens against which interventions might be deployed. Furthermore, since population-wide interventions  may be infeasible, it is important to identify which groups would yield maximal reductions in antibiotic consumption if targeted with a disease-prevention intervention. Children aged <5 years receive antibiotics at an especially high rate  and thusTo address the evidence gap in pediatric antibiotic consumption and its link with chronic comorbidities, we used medical claims data from privately insured US children to estimate the cumulative number of antibiotics dispensed by age 5, the probability of receiving at least 1 antibiotic course by age 5, and the variation among individual children in the number of antibiotic courses received. Motivated by prior studies that highlighted the important role of respiratory infections in pediatric antibiotic consumption , 10 and Data on dispensed antibiotics were obtained from pharmacy claims records aggregated in the IBM MarketScan database . These dInternational Classification of Diseases, Ninth Revision, Clinical Modification (ICD9) or revision 10 (ICD10). We restricted the sample to children continuously enrolled through age 5 who were born between 1 January 2008 and 31 December 2013 with continuous representation in both the outpatient and pharmacy claims databases, following previous methods [The database also includes outpatient and inpatient claims with a primary diagnosis coded using the Dispensed antibiotic courses were linked to the most recent outpatient visit within 7 days, following previous methods , 14. If Respiratory infection diagnoses were identified by mapping ICD codes to Clinical Classification Software (CCS) codes  and extrWe identified chronic medical conditions for each child in the cohort according to the Pediatric Medical Complexity Algorithm (PMCA) . The algWe estimated the mean cumulative number of antibiotic courses dispensed per child in the United States by age 5, the proportion of children who had received at least 1 antibiotic course by age 5, and the fraction of total antibiotic courses that were received by the top 20% of recipients. We stratified these estimates by the diagnosis that prompted them . We also estimated the change in a child's odds of being in the top 20% of antibiotic consumers as predicted by presence of a chronic condition in any of the 19 body systems described by the PMCA. For children with chronic conditions in any body systems that yielded at least a 2-fold increase in the odds of being in the top 20% of antibiotic recipients, we again measured the mean cumulative number of antibiotic courses received and the proportion of children who received at least 1 antibiotic course by age 5.To adjust for biased sampling, we weighted each claim according to the child's sex and state of residence. Weights were defined by the ratio of the number of children aged <5 years in that state/sex group in the US population according to the 2010 US census  and by tConfidence intervals (CIs) for the estimated antibiotic fill rates were calculated as exact 95% CIs for Poisson means.yi is the log-odds that individual i is in the top 20% of antibiotic consumers, Iij is an indicator that is 1 if individual i has chronic condition j and 0 otherwise, and εi is an error term. The sum is overall n = 19 body systems in the PMCA, and the βj terms are coefficients measuring the relationship between chronic conditions in each system and the odds of being in the top 20% of antibiotic consumers. The coefficient β0 is an intercept term that captures the baseline log-odds of being in the top 20% of antibiotic consumers. We estimated the coefficients using the glm function in R version 4.1.2 [P values according to a 2-tailed t-test. Full analysis code is available online [To assess the relationship between chronic medical conditions and high-volume antibiotic consumption, we conducted a categorical logistic regression to measure the change in log-odds of being in the top 20% of antibiotic consumers contributed by each chronic medical condition in the PMCA. We used the following regression model:This study was a secondary analysis of data collected for other purposes and was not considered human subjects research and not reviewed by an institutional review board.A). The mean antibiotic dispensing rate varied by age, beginning at 0.60 courses per child per year (95% CI: .59–.61) between birth and 6 months of age (180 days), then increasing to 1.9 courses per child per year (95% CI: 1.8–2.0) between 6 months (180 days) and 2 years of age (730 days), before slowing again to 1.2 courses per child per year (95% CI: 1.1–1.3) between age 2 (730 days) and age 5 (1825 days). Most antibiotic courses  were associated with respiratory infections.Children in the cohort received a mean of 6.8 (95% CI: 6.7–6.9) antibiotic courses by age 5 A. The meB).By age 5, 91% (95% CI: 90%–92%) of children had received at least 1 antibiotic course for any reason, and 85% (95% CI: 84%–86%) had received at least 1 antibiotic course for a respiratory infection B.C). The top 20% of antibiotic recipients  accounted for 52% of all dispensed antibiotic courses in this cohort.Children varied widely in the number of antibiotic courses received C. The toP < .0001), otologic conditions , and immunological conditions  were associated with the greatest increase in the odds of being in this highest-consumption group. The most common CCS codes that marked a pulmonary/respiratory, otologic, or immunological condition were related to asthma, general immunity disorders, and hearing loss, respectively . Children with a chronic pulmonary/respiratory, otologic, or immunological condition also received their first antibiotic course earlier in life: by age 1 year, 66% of children (95% CI: 65%–67%) with at least 1 such condition had received at least 1 antibiotic course vs 51% (95% CI: 50%–52%) of children without any such conditions . By age 5, 98% of children (95% CI: 96%–99%) with at least 1 such condition had received at least 1 antibiotic course vs 90% (95% CI: 89%–91%) of children without any such conditions .Children with a chronic pulmonary/respiratory, otologic, or immunological condition received more antibiotic courses  by age 5 than those without any of these conditions  and widespread (91% of children receive at least 1 antibiotic by age 5). Most  of these antibiotic courses are associated with respiratory infections. Presence of a chronic pulmonary/respiratory, otologic, or immunological condition more than double a child's risk of being among the top 20% of antibiotic recipients. Reductions in antibiotic use in this youngest age group may be possible by reducing the incidence of respiratory illnesses through vaccination or other interventions, especially among those with select chronic conditions. Such efforts should occur alongside other efforts to improve antibiotic stewardship, including improving the accuracy of diagnosis and increasing patient and physician awareness about the proper use of antibiotics.Our results are broadly consistent with prior studies that have measured cumulative antibiotic use in children in other high-income countries and, within the United States, in more geographically restricted cohorts. In New Zealand, children received a mean of 8 antibiotic prescriptions before age 5, and 97% of children received at least 1 antibiotic by age 5 . In a PhStreptococcus pneumoniae [The coronavirus disease 2019 (COVID-19) pandemic has substantially altered the transmission of and care-seeking patterns for respiratory pathogens . The timeumoniae  cases ineumoniae . This steumoniae  , since these were less likely to be represented continuously for 5 years in the data. Another limitation is that we were unable to directly observe the indication for prescribing. We inferred the indication by looking at associated diagnosis codes with recent visits proximal to the filled prescription, but this may misclassify some antibiotic courses. It is also possible that some antibiotics were linked with respiratory conditions but were not prescribed primarily for the respiratory condition; for example, if a patient was diagnosed with both bronchitis and a urinary tract infection, an antibiotic may have been given for the urinary tract infection but would have still been linked in this analysis with the respiratory condition. Additionally, we could not distinguish between appropriate and inappropriate prescribing of antibiotics or nonantibiotics. We also could not assess antibiotics that were administered directly at the point of care, so our estimates may underrepresent overall outpatient antibiotic use.In conclusion, children in the United States are exposed to antibiotics frequently and at an early age. Reducing antibiotic use remains a public health priority. Reducing rates of respiratory illness, through vaccination or other means, may be an effective way to achieve reductions in early pediatric antibiotic use, especially if these interventions are targeted to children with chronic conditions that predispose them to frequent receipt of antibiotics.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.ciac811_Supplementary_DataClick here for additional data file.


Detecting immunoglobulin G4 (IgG4)-related intracranial arteriopathy, a rare neurovascular complication of IgG4-related disease, is challenging. While magnetic resonance (MR) vessel wall imaging (VWI) can visualize various neurovascular pathologies, its application to this arteriopathy has not been reported as of this writing.A 74-year-old male and a 65-year-old female manifested multiple cranial nerve palsy and neck pain, respectively. Both cases exhibited multiorgan masses with markedly elevated serum IgG4 levels and were clinically diagnosed with IgG4-related disease. Three-dimensional T1-weighted black blood VWI with and without contrast agent identified intracranial vascular lesions characterized as nearly-circumferential mural thickening with homogeneous contrast enhancement in the internal carotid and vertebral arteries; some of the lesions had been unrecognized with screening MR angiography due to expansive remodeling. The former patient underwent corticosteroid therapy, and VWI after treatment revealed decreased mural thickening and enhancement.Further studies to elucidate characteristic findings of VWI might contribute to early detection of this treatable pathology. Immunoglobin G4-related disease (IgG4-RD) is a multiorgan immune-mediated condition . It is cWhile the aorta and coronary artery are the common vascular sites involved in this disease , only a A 74-year-old male, who complained of left facial palsy, diplopia, and dysphagia, was referred to our department. The neurological examination revealed peripheral facial palsy of House-Brackman grade III and positive curtain sign at the left soft palate; he presented with no other neurological deficit such as external ocular movement disturbance, hearing loss, limb paralysis, or ataxia. Screening MR imaging scan including time-of-flight (TOF) MR angiography had revealed infarction in the left cerebellar hemisphere and occlusion of the left vertebral artery (VA), both of which mimicked VA dissection Fig. . His neu18 F-fluorodeoxyglucose positron emission tomography integrated with computed tomography scan, revealing elevated glucose intake at and enlargement of the pancreas, kidney, and bilateral submandibular glands. Serum IgG4 level was markedly elevated (2410 mg/dL). These findings were consistent with IgG4-RD.He had a history of autoimmune pancreatitis and underwent whole-body The patient underwent 3D T1-weighted black blood VWI with and without contrast agent using a 3 tesla MR unit  with 32-channel head coil. The imaging parameters were as follows: TR = 1000 ms, TE = 11 ms, field of view = 180 × 180 mm, matrix = 320 × 320, slice thickness = 0.56 mm (0.56 mm isotropic voxel), bandwidth = 380 Hz/pixel, acceleration factor = 2 × 2, and scan time = 5 m 34 s. To improve imaging contrast between the vessel wall and cerebrospinal fluid, the imaging sequence was prepared by DANTE prepulse –15. The VWI revealed lesions not only at the left intracranial VA but also at the supra-clinoid portion of the bilateral internal carotid arteries (ICAs) Fig. . The latT1-weighted contrast enhanced MR imaging scan had also revealed enhanced mass lesions at the bilateral lacrimal glands and at the geniculate ganglion of the left facial nerve. The patient’s neurological symptoms were attributable to these lesions and/or direct inflammation spreading from the arteritis to the cranial nerves. Antiplatelet agent was not administered. He did not undergo biopsy of the intracranial vessel wall because the procedure is highly invasive.He was referred to immunologists, and corticosteroid treatment (prednisolone 40 mg per day) was started. This treatment was tolerated and no adverse event occurred. His symptoms were resolved shortly after the treatment. Serum IgG4 level was significantly reduced (185 mg/dL) 4 months after treatment initiation. VWI performed at the same period revealed decreased mural thickening and enhancement in the bilateral supra-clinoid ICAs Fig. E. A simiA 65-year-old female complained of pain at the neck, scapula, and left arm. She was treated with analgesia by a general physician; however, the pain gradually worsened, and she was referred to our hospital.Screening cervical CT scan revealed a tumor mass progressing from the intervertebral foramen to the left brachial plexus. It also incidentally revealed bilateral lacrimal gland enlargement and an unidentified enhanced lesion around the left VA. Serum IgG4 level was elevated (484 mg/dL). TOF MR angiography detected only minimal luminal irregularity at the left VA Fig.  A. On thDANTE-prepared 3D T1-weighted black blood VWI revealed circumferential massive mural thickening with homogeneous contrast enhancement at the left VA Fig.  A and B,The present two cases shared a unique finding on VWI: nearly-circumferential mural thickening with homogeneous contrast enhancement. Some lesions had not been recognized with screening TOF MR angiography because expansive remodeling kept the lumen almost intact. The lesions decreased shortly after corticosteroid treatment; this reasonably suggests that there was an association between these lesions and IgG4-RD. As of this writing, this is the first report describing the VWI finding of IgG4-related intracranial arteriopathy.Case 1) [The present cases are consistent with the literature on IgG4-related intracranial arteriopathy, which exhibits various stenotic and aneurysmal formations. Kondo et al. and Ikeoka et al. reported similar cases, both of which exhibited stenosis of the middle cerebral artery causing infarct , 10. On Case 1) . They foThe finding of VWI corresponds to the histopathology of IgG4-related arteritis occurring in the coronary artery and aorta. Matsumoto et al. described a case with coronary arteritis, whose histopathology showed circumferential mural thickening with massive infiltration of inflammatory cells including IgG4-positive plasmacytes . They alVWI with and without contrast agent might facilitate the early detection and differential diagnosis of IgG4-related intracranial arteriopathy, both of which might be challenging with other imaging modalities. Early detection of the pathology is particularly important because it causes fatal sequelae , 8 but m