Perfluorinated acids(PFAs) are widely used synthetic chemicalcompounds, highly resistant to environmental degradation. The widespreadPFA contamination in remote regions such as the High Arctic impliescurrently not understood long-range atmospheric transport pathways.Here, we report that perfluorooctanoic acid (PFOA) initiates heterogeneousice nucleation at temperatures as high as −16 °C. In contrast,the eight-carbon octanoic acid, perfluorooctanesulfonic acid, anddeprotonated PFOA showed poor ice nucleating capabilities. The icenucleation ability of PFOA correlates with the formation of a PFOAmonolayer at the air–water interface, suggesting a mechanismin which the aligned hydroxyl groups of the carboxylic acid moietiesprovide a lattice matching to ice. The ice nucleation capabilitiesof fluorinated compounds like PFOA might be relevant for cloud glaciationin the atmosphere and the removal of these persistent pollutants bywet deposition. Of the perfluorinated acids, PFOA is the most ubiquitouspollutant due to its extensive usage in the fluoropolymer industryand high total emissions.5 PFOA has been observedin different air and water sources  and was detectedin regions as remote as the High Arctic.7 Since thereare no primary sources of PFOA in remote locations that could contributeto contamination, questions arise regarding the sources and transportpathways of this concerning pollutant.8 The currentlysuggested long-range transport pathways of PFOAs are hydrosphericand atmospheric, with the latter being more relevant for remote locationsand the Arctic.10 This can be witnessed by highPFA and PFOA concentrations in the Arctic atmosphere and ongoing detectionof PFOA and PFAs in Arctic snow samples.11Perfluorinated acids (PFAs)such as perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid(PFOS) are anthropogenically generated compounds that have emergedas significant global environmental pollutants with persistent, bioaccumulative,and toxic properties.12 but they could also interact with clouds,13 which are important for weather effects due to cloud glaciationand precipitation. Pure water droplets do not freeze homogeneouslyuntil ∼ −38 °C owing to the energy barrier associatedwith creating the initial crystallization nucleus.14 In cloud droplets, water typically freezes in a heterogeneousprocess, facilitated by the presence of particles that serve as icenucleators (IN). Common abiotic IN include clay, dust, minerals, orcarbonaceous materials.15 Biogenic IN consistof biomolecules derived from bacteria, fungi, insects, or pollen.16 Among the abiotic ice-nucleating surfaces, monolayersof long-chain alcohols have been shown to be particularly effective,while fatty acids with similar chain lengths are significantly lessso.17 Here, we report that PFOA is an efficientIN, much more so than the structurally similar PFOS and octanoic acid(OA). These compounds consist of a hydrophobic tail and a hydrophilicheadgroup, and are known to accumulate and form monolayers at theair–water interface (18 The ice nucleationactivities of the (fluoro)surfactants are investigated using the high-throughputTwin-plate Ice Nucleation Assay (TINA).19 TINA enables the simultaneous measurement of a complete dilutionseries with each series composed of hundreds of droplets of a fewmicroliters with high statistics, enabling the analysis and characterizationof the efficiency of particles with high accuracy.22Withinthe atmosphere, perfluorinated compounds can undergo atmosphericoxidation and react with Criegee intermediates,nterface 1.18 The T50 values of PFOA solutions as a function of concentration.The T50 values are defined as the temperaturesat which 50% of the droplets are frozen. PFOA shows considerable icenucleating activity, in a manner highly dependent on the solutionconcentration.T50 = ∼ −28 °C, comparable to pure water in our experimentalsetup. Increasing the concentration above 0.02 mg/L results in freezingtemperatures that are higher than that of pure water. We find thatfor 200 mg/L PFOA solutions T50 = ∼−21 °C, and for 2000 mg/L solutions, T50 increases up to ∼ −16 °C. Whilethe maximal determined T50 value is ∼−16 °C, it is also worth mentioning that we occasionallyobserved high initial freezing temperatures of up to −5 °Ceven at low concentrations surfactants. For OA,we found that the maximal ice nucleation activity is at ∼ −24°C, which is only slightly higher than the freezing temperatureof pure water in our setup. Apparently, the perhydrogenated fattyacid is a significantly poorer ice nucleator than perfluorinated PFOA. For PFOS, the maximal icenucleation activity was ∼ −20.5 °C, but at 20 timeshigher concentration than PFOA. Deprotonation of the carboxylic acidheadgroup of PFOA eliminates most ice nucleation activity with a maximumof ∼ −26.5 °C. It seems that both changing thehydrophilic headgroup of PFOA or the hydrophobicity of the tail suppressesthe ice nucleation activities of the respective (fluoro)surfactants.We performed dynamic light scattering and calorimetric measurementsto examine whether different water activities or solution aggregatesmay be the origin of the observed differences in the ice nucleationcapabilities. Neither the melting points of the compounds nor theiraggregate sizes were found to differ notably , eliminatingexplanations involving different water activities or aggregate sizesin solution as the origins for the observed differences in ice nucleationactivity.18 with a maximum surface excess of ∼2 mg/m2 foraqueous concentrations exceeding 100 mg/L. In the TINA droplet freezingexperiments, the surface pressure cannot be controlled and is a functionof the amount of PFOA at the surface and the temperature. Interestingly,we find that the observed ice nucleation activities of PFOA and theother surfactants directly correlate with their surface tensions,implying that their ice nucleation activities are linked to the buildupof the (fluoro)surfactant monolayers .We exclude the possibility that multilayered structures or micellesform or coexist underneath the PFOA monolayer, since we observe nochanges in aggregate size in DLS measurements . The critical micelle concentration for PFOA alsofalls below the solubility limit, and X-ray reflectivity measurementsshowed that the thickness of a perfluorinated carboxylic acid layercorresponds to a monolayer state of the film.23PFOA and other fluorosurfactantsare known to accumulate and formmonolayers at the air–water interface,n-alkylalcohols havebeen shown to be particularly effective in nucleating ice and thattheir freezing temperatures increased with the length of the hydrocarbontail.17 These monolayers expose hydroxylgroups to water in a manner that resembles the basal plane of ice.Hence, it was suggested that the structural lattice matching withice governs their ice-nucleating efficiency. Interestingly, fattyacid monolayers, which also expose hydroxyl groups to water, are verypoor ice nucleators, with solid fatty acid crystals showing more promise.24 Molecular simulations have previously suggestedthat the discrepancy between the alcohol and fatty acid layers wasdue to differences in the monolayer’s compactness and the resultingstructural match to ice, which are key for determining the ice nucleationability of organic surfaces that expose hydroxyl groups to ice.25Previously, monolayers of 2) than hydrogenated chains (∼0.19 nm2) and thus lower interfacial densities and molar volumes than hydrogenatedchains with the same number of carbon atoms.27 There are also conformational differences. For perfluorinated chains,the dihedral angle at minimum energy is not exactly 180°, asit is for hydrogenated ones. Consequently, perfluorinated chains adopta characteristic helical conformation, while hydrogenated chains tendto be in an all-trans planar zigzag form.30 Moreover, the energy barrier for internal rotation of perfluorinatedchains is appreciably higher than for hydrogenated chains, which inducea rigid character, in contrast with the flexible character of hydrogenatedchains.29Upon fluorination, hydrophobic chainswill undergo structural andconformational changes that directly affect the packing of the monolayer.Structurally, perfluorinated chains display a larger footprint . Our conclusion is also in line with previous suggestions basedon MD simulations and experimental findings that solid fatty acidparticles are better INs than fatty acid monolayers.25 Irrespective of the precise molecular mechanism, the finding thatPFAs have high ice nucleation activity may have direct implicationsfor the transport and environmental fate of these persistent organicpollutants, as they could get distributed to remote environments byactively being involved in cloud glaciation.We conclude that upon fluorination, the morphology and packing within the monolayerallow for a better alignment of the carboxylic acid groups with lessstructural fluctuations, thereby providing a better ice template andenabling enhanced nucleation properties. This hypothesis is supportedby additional measurements of perfluorodecanoic acid, which also showsgood ice nucleation abilities (3),32 and the deprotonated PFOA form is likely prevalent, whichhas low ice nucleation activity.33 Ourresults suggest that increasing the crystallinity and order of monolayersthrough perfluorination will also affect the ice nucleation abilitiesof other perfluorinated compounds such as long-chain alcohols, potentiallyrendering them from good to exceptional ice nucleators with directatmospheric implications.17 The possible ice nucleationsynergy between hydrogenated and perfluorinated long-chain alcoholsand the interplay of PFAs with other ice-nucleating particles foundin the atmosphere are yet to be investigated.While the localconcentration at the anthropogenic point of originmay be high, once it becomes distributed in the environment, averageconcentrations found in the atmosphere are significantly lower thanthe ones reported here (∼15 pg/m


Measurements of allergen-specific IgE antibodies with different manufacturers’ assays show modest or poor agreement. This study compares analytical performance of specific IgE tests for whole allergen extracts and individual allergen components of two assay systems, IMMULITE and ImmunoCAP, using human sera as well as monoclonal antibodies.Comparisons were performed for specific IgE to house dust mite , egg white  and the allergen components rDer p 1, rDer p 2, nGal d 1, nGal d 2 and nGal d 4 in human sera and with monoclonal mouse/human chimeric IgE antibodies specific for the same allergen components. Competitive interference with IgE measurement was investigated using allergen-specific monoclonal IgG and IgG4 antibodies.Measurements of IgE to HDM and EW in serial dilutions of human sera revealed weaker dilution linearity with IMMULITE than with ImmunoCAP. Analysis of five different monoclonal IgE antibodies with total and specific IgE assays, expected to return similar levels, gave an average specific/total IgE ratio of 0.96 (range 0.71–1.14) with ImmunoCAP and 1.89 (range 0.76–2.85) with IMMULITE, indicating overestimation of specific IgE by IMMULITE. With the EW IgE tests of both assay systems, measurements of a chimeric anti-Gal d 2 IgE antibody were unaffected by a competing mouse IgG antibody. While the same was true for measurement of a chimeric anti-Der p 1 IgE antibody using the HDM test in ImmunoCAP, a suppression of measured concentrations by up to 42% was observed in IMMULITE. Similarly, measurement of HDM-specific IgE in human sera by ImmunoCAP was unaffected by a competing monoclonal anti-Der p 2 IgG4 antibody while IMMULITE displayed a reduction of HDM-specific IgE values by up to 30%.In this evaluation of analytical performance of two widely used assay systems, ImmunoCAP showed higher accuracy in quantitation of specific IgE and greater resistance against competing allergen-specific non-IgE antibodies which may arise through natural or dietary exposure, or as a result of allergen immunotherapy treatment. House dust mite (HDM) allergy has a significant clinical effect on lung function in children . Among pAs far as food allergy is concerned, egg white is one of the most common causes in children. Prevalence figures of 0.5–2.5% have been reported  and its A/L (ImmunoCAP) failed an egg OFC while 95% of those with a level less than 0.9 kUA/L passed an egg OFC. Similar results were reported by Ando et al. [A/L and 1.6 kUA/L, respectively. Thus, when measuring Gal d 1-specific IgE antibodies, high test precision is clinically important, not only in the high concentration region but also at low concentrations. In addition to Gal d 1, there are other important EW components such as ovalbumin  and lysozyme .Allergic reactions can occur either to any form of egg or, in some patients, mainly to raw or lightly cooked / baked egg , 14. Clio et al. , defininIt is of utmost importance in sIgE testing that measurements are truly specific and accurate. Currently, there are three commercially available specific IgE immunoassay platforms in Japan, of which IMMULITE® 2000  and ImmunoCAP™  are dominant. Both platforms provide analysis of IgE to a wide range of allergens, including crude HDM and EW extracts and the HDM and EW allergen components Der p 1 and Der p 2 and Gal d 1, Gal d 2 and Gal d 4, respectively. Earlier comparisons of the two systems have revealed both significant differences in test results as well as similar performances –19. The Gradually declining levels of egg specific IgE and milder symptoms on accidental exposure or in food challenge are signs of tolerance development. At the age of 16 years, around 70% of egg allergic children will have developed tolerance .Forty-four HDM-positive and 36 EW-positive serum samples were included in the study. For the EW, samples were selected from a patient pool where all patients had been diagnosed as having egg allergy while for the HDM, samples were selected from sensitized patients but with no access to their clinical diagnosis. Of each sample a series of three dilutions  was prepared using the ImmunoCAP IgE/ECP/Tryptase Sample Diluent. Hence, including undiluted specimens, all samples were available at four different concentrations for the assay comparisons. In a dilution linearity study, the observed (O) IgE concentration measured in each sample dilution point was evaluated against the expected (E) value derived from the measured IgE concentration in the undiluted sample, divided by the dilution factor, as an O/E ratio. For each of HDM and EW, an average O/E ratio was calculated from the O/E ratios of all dilution measurements. Weighted linear regression lines were fitted to the data with either ImmunoCAP or IMMULITE ratios as the dependent variables and ImmunoCAP / IMMULITE initial sIgE values as the independent variables.Five allergen-specific mouse-human chimeric monoclonal IgE antibodies were also employed in the evaluation, each consisting of an allergen-specific variable domain from a mouse monoclonal antibody and the constant part of the heavy chain of human IgE. The antibodies reacted specifically with Der p 1, Der p 2, Gal d 1, Gal d 2 and Gal d 4, respectively, and could be measured with the specific and total IgE assays of both assay platforms. Furthermore, for each Ab a 6-step dilution series was prepared, up to a dilution factor of 16. Hence, samples with seven different concentrations of each Ab were available for the assay comparisons. The chimeric IgE antibodies to Der p 1, Gal d 1, Gal d 2 and Gal d 4 were obtained from Thermo Fisher Scientific  while the chimeric IgE antibody against Der p 2 was obtained from Indoor Biotechnologies .Competition experiments were carried out using mouse monoclonal IgG antibodies against Der p 1 and Gal d 2 (Thermo Fisher Scientific) and a mouse/human IgG4 antibody against Der p 2 (Indoor Biotechnologies). Each mouse monoclonal antibody was added at three different concentrations  to the anti-Der p 1 or anti-Gal d 2 chimeric IgE antibody samples. Analysis was performed with specific IgE assays for HDM and EW, respectively. The results were evaluated as ratios to a control to which buffer alone had been added instead of the competing IgG antibody.A/L, respectively), followed by measurement of HDM-specific IgE. The results were evaluated as ratios to a control to which buffer alone had been added. The concentration of Der p 2 specific IgG4 antibody was determined using the ImmunoCAP specific IgG4 assay (Thermo Fisher Scientific). A total of 4 comparative studies were thus performed in this work, summarized in Table The anti-Der p 2 chimeric IgG4 antibody was added at three different concentrations  to two HDM-positive serum samples  kU/L for HDM and 58.9–481  kU/L for EW  and the EW sIgE values between 15.8 and 98.6  kUThe ImmunoCAP assays displayed dilution linearity with average O/E ratios of 1.21  and 1.26  for HDM and EW, respectively. The IMMULITE assays displayed dilution linearity with average O/E ratios of 1.29  and 1.44  for HDM and EW, respectively. Weighted linear regression lines were fitted to the data as shown in Fig. Compared with ImmunoCAP, IMMULITE HDM showed greater range of variation in the O/E results (p < 0.001). As demonstrated, the IMMULITE HDM assay ratios increased with increasing levels of sIgE. For both ImmunoCAP and IMMULITE EW, the variation ranges in the O/E result were quite similar while the mean value of the O/E ratio was higher for IMMULITE EW (p < 0.001).In a sample of a monoclonal IgE antibody, where all IgE is represented by a single allergen specificity, measurements of total and specific IgE should yield comparable concentration values. With ImmunoCAP, the levels of sIgE to Der p 1, Der p 2, Gal d 2 and Gal d 4 measured in all 6 dilutions of the chimeric IgE antibodies were comparable to the total IgE levels measured in the same antibody samples, with an average sIgE/tIgE ratio close to 1 (range 0.92–1.14)  in IMMULITE.A robust assay is characterized by its ability to deliver consistent results from specimens regardless of their content of potential interferents. One important cause of potential interference is allergen specific non-IgE antibodies which may be induced by natural allergen exposure or allergen immunotherapy (AIT) treatment. In this study, we assessed the resilience of the ImmunoCAP and IMMULITE HDM and EW IgE assays against such interference by competing with the increasing concentrations of monoclonal IgG antibodies of the same allergen specificity prior to IgE measurement. The results of this experiment are shown in Fig. While the above competition experiment using chimeric IgE antibodies addressed the IgE binding capacity of the assays in an analytical set-up, we also studied their ability to withstand competing antibodies in a situation more closely reflecting their clinical use. This was done by assessing the effect on HDM IgE measurements in patient samples by increasing concentrations of a chimeric Der p 2 specific IgG4 antibody. The results of this experiment are shown in Fig. In this study, the ability of the ImmunoCAP and IMMULITE IgE assay systems to accurately quantify allergen specific IgE antibodies was studied. Main findings were generally poor agreement between the two systems in the measurement of specific IgE levels to HDM and EW. This is especially true for the results in the linearity study where the effect in IMMULITE HDM of the serial dilutions on sIgE recovery was more variable compared to ImmunoCAP. Furthermore, for IMMULITE EW, mean sIgE results from the diluted samples was higher than for the results with ImmunoCAP (1.44 vs 1.29). In a perfect linearity dilution study, the mean O/E ratio should be 1.Differences in assay performance between the two systems were further investigated using chimeric IgE antibodies. In a sample where all IgE antibodies are represented by a single allergen specificity, measurements of total and specific IgE should yield comparable concentration values. Since a close agreement of total IgE concentrations measured with the IMMULITE and ImmunoCAP systems has earlier been demonstrated  and bothThe two interference studies performed showed that IgE measurements with the ImmunoCAP HDM and EW IgE assays were unaffected by the addition of the competing IgG or IgG4 antibodies, as was the IMMULITE EW IgE assay. In contrast, significant interference was observed with the IMMULITE HDM IgE assay, both when the chimeric anti-Der p 1 IgE antibody and human patient samples were measured in competition with anti-Der p 1 IgG and anti-Der p 2 IgG4, respectively, with a reduction of measured specific IgE values by up to 42%. The important implication of the observed interference is that levels of IgE to HDM will not be accurately measured in patients who have mounted an IgG response against HDM, such as during HDM immunotherapy.Taken together, this study demonstrates significant differences in performance of specific IgE assays between the two assay systems with respect to quantitative accuracy. Additionally, different resistance to interference by competing IgG antibodies was demonstrated which may affect IgE measurement in patients treated with allergen immunotherapy. The findings highlight that despite the use of the same concentration units of specific IgE and calibration against the WHO reference preparation of IgE, different test results for a patient sample may be obtained depending on the assay system used by the laboratory. As a consequence, different diagnostic conclusions may be drawn, affecting the management of the patient.A limitation in this study is the relatively small number of sera in the dilution linearity part, with 44 and 36 sera for the HDM and EW group, respectively. A study with a greater number of sera, spread evenly over the entire measuring range, may have provided for a more robust and representative comparison of the assay systems.This study demonstrates significant differences in analytical performance of two specific IgE assay systems for whole extracts and individual allergen components. The results indicate higher accuracy of quantitation of specific IgE and a higher resistance to competing allergen-specific non-IgE antibodies of ImmunoCAP as compared to IMMULITE.


Analysis of variance F statistic for the effect of NPWT over 180 days was highly significant . There were no statistically significant differences between the time to first CRI and PD-related peritonitis between the two groups. There was one case of CRI with relapsing peritonitis and catheter loss in the non-NPWT group. These findings demonstrate the association between NPWT and low exit-site score. NPWT can be recommended for the management of PD catheter exit sites in the early postoperative period.Peritoneal dialysis (PD) catheter exit-site care is critically important for the prevention of catheter-related infections (CRIs) and subsequent peritonitis. The postoperative management of the site is particularly essential because it has an open wound that is always adjacent to a PD catheter tube. This study aimed to examine the effectiveness of negative-pressure wound therapy (NPWT) for postoperative PD catheter exit sites. Thirty patients with end-stage renal disease who underwent simultaneous PD catheter insertion and exit-site formation were randomly assigned to receive NPWT (NPWT group) or conventional dressing (non-NPWT group) for the first seven postoperative days. The exit-site scores on the seventh postoperative day was lower in the NPWT group than in the non-NPWT group ( It is reported to be a direct or major contributing cause of death in about 16% of PD patients3. Moreover, peritonitis is a major cause of discontinuation of PD and change to long-term hemodialysis2. Exit-site infections (ESIs) and catheter-tunnel infections, which are usually described as catheter-related infections (CRIs), are major predisposing factors of PD-related peritonitis5. National and international consensus guidelines, which are issued by the International Society for Peritoneal Dialysis (ISPD), focus on identifying the best strategies for the prevention and treatment of CRIs and PD-related peritonitis6. Many of the guidelines, which provide strong recommendations, are controversial or supported by limited evidence. However, it is clear from the guidelines that PD catheter exit-site care is critically important for the prevention of CRIs and PD-related peritonitis.Peritoneal dialysis (PD)-related peritonitis is one of the most common and most serious complications of PD8, is a noninvasive wound healing technique used for a wide range of complex wounds9. NPWT involves the delivery of partial vacuum with suction through a special dressing and therapy unit, which creates an environment that promotes wound healing. In current clinical settings, NPWT is used for a variety of wound types, including open traumatic wounds10, skin grafts11, burns12, venous leg ulcers13, diabetic foot ulcers14, and surgical site infections15.A PD catheter exit site is an open wound made during operation using a tunneler, and it is always in contact with a catheter tube. Retardation of exit-site healing in the postoperative period sometimes leads to early CRIs. Negative-pressure wound therapy (NPWT), first developed in the early 1990s. suggested that NPWT is effective for PD catheter exit sites16; however, this was an observational study. The present study aimed to evaluate the effectiveness of NPWT for postoperative PD catheter exit-site care via a randomized open-label trial.A study by Mori et alFor this study, 40 patients were assessed for eligibility Fig. . Ten patp = 0.0049, Mann–Whitney U test). Regarding the secondary outcomes, analysis of variance revealed that exit-site score over 180 days after the operation was also statistically lower in the NPWT group than in the non-NPWT group (p = 0.007). There were no statistically significant differences in time to first CRI and PD-related peritonitis between the two groups .The procedure outcomes are summarized in Figs. In the non-NPWT group, there was a case of catheter loss due to PD-related infections. The patient presented with ESI a month after operation and had relapsing PD-related peritonitis, which warranted the removal and reinsertion of the PD catheter.A PD catheter exit site is an open wound made using a tunneler, and it is always adjacent to a catheter tube. Catheters are typically made of smooth silicone rubber, and prolonged exit-site healing sometimes leads to early CRIs. Hence, strategies that prevent delay of exit-site healing and promote exit-site stabilization are essential for PD patients.18. The concept provides clinicians with a comprehensive approach to removing barriers to healing and stimulating the healing process for maximum benefit. Schultz et al. proposed the TIME acronym, which highlights four elements that need to be resolved with regard to wound bed preparation19. “T” stands for tissue, “I” stands for inflammation and infection, “M” refers to moisture imbalance, and “E” refers to epithelial edge advancement. According to this concept, adequate management of the postoperative exit site of a PD catheter is necessary to avoid infection, moisture imbalance, and advancement of the wound edges.Wound bed preparation is a systematic strategy aimed at improving wound management by identifying and removing barriers to wound healing. The prototype of the concept emerged in the 1990s, gradually gained recognition, and developed into a systematized paradigm for the strategy of chronic wound management. hypothesized that loose fit of skin around the PD catheter would increase the risk of trauma and contamination20. NPWT can reduce the surface area of the open wound and lead to early tight fixation. We posit that these three principal mechanisms discussed above great influence the PD catheter exit site as evidenced by the significantly low exit-site scores and the early stabilization of the exit site in the NPWT group. In contrast, the non-NPWT group tended to have high exit-site scores on the seventh postoperative day as evidenced by redness, crusts, and serous drainage. This finding suggests that the exit site is susceptible to trauma due to loose fixation and moisture imbalance caused by the extra exudate, especially after the operation. These factors also paradoxically reveal the usefulness of NPWT for postoperative exit sites.In our study, the NPWT group had lower exit-site scores on the seventh postoperative day than the non-NPWT group. The proposed effects of NPWT on postoperative PD catheter exit sites are discussed below. First, the sites are kept hygienic because they are covered with special foam dressings and sealed with clear drape on the operative field. Second, continuous negative pressure removes exudate, resulting in a properly moisturized environment. The PD catheter is blindly directed through a subcutaneous tunnel to the marked site using a tunneler. This procedure frequently causes injury to small vessels in the subcutaneous tissue. In such cases of injury, prolonged bleeding from the exit site often occurs. The system of vacuum with suction is also effective for eliminating infectious wastes at the exit site. Third, the negative pressure causes contraction of the wound edge. Twadroski et al21. The study found that patient adherence to proper hand hygiene, mask wearing, exit-site observation, secretion examination, and effective communication with PD staff influenced CRI history and exit-site issues. Russo et al. reported that approximately one-third of PD patients needed reinforcements in knowledge and ability to correctly perform PD-related infection control and concluded the retraining needs of patients on PD22. As described, educating PD patients to recognize the importance of exit-site assessment, daily self-care, and retraining of the relevant skills must be considered useful for preventing CRIs.Regarding secondary outcomes, the NPWT group also had lower exit-site scores over 180 days than the non-NPWT group. However, no statistically significant differences in time to first CRI and PD-related peritonitis were noted between the two groups. These results suggest that, over the long term, exit-site score and the incidence of CRI are independent variables. We hypothesized that daily self-care practice on the exit site has a major impact on long-term CRI and PD catheter-related peritonitis. Although our center ensured that patients underwent a three-week PD training conducted by nursing staff with the appropriate qualifications and experience during their hospitalization, daily PD catheter exit-site care tends to become more personalized as the PD duration increases. Ding et al. reported that the level of adherence to different aspects of exit-site care varies among PD patients24. Although PD catheter exit site “down-growth” has been reported in a few studies, only the study by Yoshino et al. reported that redundant “down-growth” and increase in dead space are associated with insufficient catheter fixation and altered metabolism25. It is easy to anticipate that trauma and contamination in the sinus directly increase the risks of CRIs and subsequent peritonitis.“Down-growth” may also contribute to the long-term acute outbreak of infections. As earlier noted, a PD catheter exit site is an open wound that is always adjacent to a catheter tube. Resistance to infection depends on the formation of a biological barrier formed from tissue growing along a subcutaneous cuff made of polyethylene terephthalate. The epithelium contiguous to the silicone catheter tends to invert toward the cuff, creating a sinus between the tube and the skin. The exit site on which the “down-growth” forms under experimental conditions tends not to have swelling, crust, redness, or pain unless it is infected. This is because a biological barrier composed of epithelium typically covers the catheter tube completely. However, the “down-growth” forms a dead space that promotes accumulation of metabolic products and bacterial colonization, which result in CRIs26. Using this scoring, ESI is diagnosed via ≥ 4 points or through the presence of purulent discharge with or without skin erythema at the catheter–epidermal interface. There has been uncertainty regarding the usefulness of this scoring system, as observed in some recent studies28. Furthermore, regularly monitoring exit-site score itself cannot predict or prevent CRIs. In contrast, Sangeetha et al.29 previously reported that patients with an ISPD exit-site score of 3 points fell in the category of “equivocal” under the Twardowski and Prowant’s exit-site classification and developed peritonitis in 27.2% with catheter loss in all of them, implying that clinicians should be more vigilant about the possibility of developing infection as the score is higher even though it is within 3 points. This reiterates that the scoring system is a valuable scale, and in our trial, it is considered the most universal and objective measurement for verifying the effectiveness of NPWT for postoperative PD catheter exit site. Moreover, maintaining a low exit-site score, i.e., being categorized as “perfect” or “good” under the Twardowski and Prowant’s exit-site classification reflects a CRI-free condition, which may prevent PD-related peritonitis.The exit-site scoring system provided by ISPD was originally proposed by Schaefer et al. and applied to pediatric patientsThe cost of NPWT is USD 90 per day. The high cost may hinder its wide application to a PD catheter exit site. Therefore, it is arguable whether NPWT is a cost-effective therapy for the management of postoperative PD catheter exit site.16; however, the study was observational. Our randomized prospective study shows that NPWT is associated with low exit-site scores in the early postoperative period and early stabilization of the exit site.To the best of our knowledge, this study is the first randomized open-label trial to examine the effectiveness of NPWT for postoperative PD catheter exit sites. Over the years, many strategies aimed at preventing CRI and subsequent peritonitis have been attempted. Although ISPD guidelines provide some strong recommendations, many of them are controversial or supported by limited evidence. In the study by Mori et al., it was reported that NPWT is effective for early tight fixation to the skin around the catheter30.Our study has some limitations that should be noted. The required sample size of this study was calculated based on the estimation of statistically significant difference in exit-site scores. Although our study did not reveal statistically significant differences in time to first CRI and PD-related peritonitis between the NPWT and non-NPWT groups, larger samples and longer follow-up durations are necessary for more accurate assessments of PD patients. This is because the incidences of CRI and PD-related peritonitis have decreased considerably; for example, the incidence rate of PD-related peritonitis was reported to be 0.26 episodes/patient-yearIn conclusion, this study is the first randomized open-label trial to assess the effectiveness of NPWT for postoperative exit sites by comparing NPWT with conventional dressing in terms of exit-site score. The NPWT group had significantly lower exit-site scores on the seventh postoperative day and over 180 days than the non-NPWT group. Therefore, we recommend NPWT as an option for early stabilization of the exit site.This study was a single-center, randomized, open-label study conducted from November 2017 to April 2021 at Juntendo University Hospital, Tokyo, Japan. The study protocol was approved by the Ethics Committee of Juntendo University Hospital, Tokyo, Japan . In the present study, all procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All participants provided written informed consent before enrollment. The study was registered with University Hospital Medical Information Network Clinical Trials Registry on 19/11/2017 (UMIN000030033).31.Patients scheduled to undergo PD catheter insertion surgery were enrolled in the study. The inclusion criteria were as follows: (1) age ≥ 20 years, (2) planned simultaneous PD catheter insertion and exit-site formation using a tunneler from November 2017 to November 2020, (3) exit-site creation on the abdominal wall, and (4) assumed ability to perform daily exit-site care. The exclusion criteria were as follows: (1) poor general status, (2) refusal to participate in the study, (3) perceived ineligibility, and (4) PD catheter implantation with stepwise initiation of PD using the Moncrief–Popovich technique, which entails the implantation of a catheter, with its end buried in subcutaneous tissue, until the initiation of PDEligible patients for the trial were randomized via a simple computer-generated randomization chart into those who would undergo NPWT (NPWT group) or conventional dressings (non-NPWT group) of the exit site Fig. .The proposed exit site on the abdominal wall was examined preoperatively with the patient lying down, standing up, and sitting up. The exit site was also checked to ensure that it is sufficiently visible for easy daily exit-site care and that there is no interference with skin wrinkles or the belt line.The PD catheter insertion surgery was performed under general anesthesia. To prevent surgical site infection, all patients received 1 g cefazolin 1 h before the surgical incision was made. Under direct view, a 5-cm vertical paramedian incision was made 2 cm below the umbilicus and 2 cm to the right or left of the midline. The underlying subcutaneous tissues were opened until the anterior rectus sheath was exposed. The muscle fibers were bluntly separated to reach the posterior rectus sheath. The fascia of the posterior rectus sheath and the peritoneum were lifted and incised to enter the abdominal cavity. A straight PD catheter with double cuffs (Long Shoot Catheter 650 with reinforcement JL-1(A)S®; Hayashidera Medinol, Kanazawa, Japan) was inserted with an internal stiffening stylet. The internal cuff was fixed to the posterior sheath and the peritoneum with 2–0 coated braided polyester sutures  after testing flow function by injecting 100 mL normal saline solution. To prevent fluid leakage, a strong purse-string suture was made at the level of the posterior muscle and the peritoneum. The PD catheter was then directed through a subcutaneous tunnel to the previously marked skin exit site using a tunneler. All skin exit sites were directed downward or laterally (not upward). Confirmation of successful fill and drain of 1000 mL dialysate was followed by closure of the vertical paramedian incision. All patients were administered 2 g cefazolin daily for 3 days after PD catheter insertion.Patients assigned to the NPWT group received treatment delivered through the vacuum-assisted closure device  immediately after the operation. The exit site was covered with special foam dressings  and sealed with a clear drape  that helped maintain negative pressure over the site. One end of the tube connected to the dressing through the pressure-sensing pad , and the other end connected to the VAC® Therapy Unit and Canister system (KCI). Once connected to the device, a continuous negative pressure of 125 mmHg was applied in accordance with the instruction manual. NPWT was administered at the exit sites, and the dressing was left in situ for 7 days until the patients underwent first assessment of the site.Patients assigned to standard care (the non-NPWT group) were treated using waterproof see-through film dressings. When filled with exudate, the dressings were changed appropriately.5. The patients were taught to begin exit-site care and fluid exchange with hand washing and mask wearing. After examining the sites by themselves, the patients were trained to clean each site with a cotton swab soaked in 0.05% chlorhexidine and to cover with sterile gauze. The PD catheter was properly fixed to the abdominal wall by gentle taping onto the skin to avoid traction, which could cause injury and bleeding.Over a three-week hospitalization period, the patients underwent PD training conducted by nursing staff with appropriate qualifications and experience in accordance with the ISPD guidelinesAfter discharge, the patients underwent monthly assessment of the exit site conducted by the attending doctor and nursing staff. The exit site was also photographed for further evaluation. The patients were followed up for 180 days from the day of PD catheter insertion.The primary outcome of the present study was exit-site score on the seventh postoperative day. The secondary outcomes were exit-site score over 180 days, time to first CRI, time to first PD-related peritonitis, and catheter loss due to PD-related infections.5. The exit-site scoring system, which was first proposed by Schaefer et al.  with a white blood cell count ≥ 100/μL, comprising ≥ 50% polymorphonuclear cells1.The exit-site score was calculated according to the ISPD guidelinesl. Table 26, is reThe sample size estimate was calculated for the primary outcome of exit-site score on the seventh postoperative day, by nonparametric analysis using the wmwpow package. In the non-NPWT group, the rates of patients with exit-site scores of 0, 1, and 2 were 0%, 50%, and 50%, respectively. In contrast, in the NPWT group, the rates of patients with exit-site scores of 0, 1, and 2 were 30%, 60%, and 10%, respectively. In total, 15 patients were recruited for each group after accounting one patient for loss to follow-up. With an alpha error of 0.05 and a beta error of 0.2 (80% power), the required sample size was 14 patients in each group.U and Fisher’s exact tests were used to compare baseline quantitative and categorical variables, respectively, between the two groups. Data were presented as medians  unless otherwise stated. The primary endpoint was analyzed using the Mann–Whitney U test, analysis of variance was used for all observational periods of the exit-site score, and the Kaplan–Meier analysis was used for event-free analysis.The NPWT group was compared to the non-NPWT group. The Mann–Whitney Statistical analyses were performed using SAS software  and R statistical software package (R version 4.0.3). R package is an open-source environment for statistical data analysis, and it is readily available . A p value of < 0.05 was considered to indicate statistical significance.


A 54‐year‐old man developed a severe anaphylactic penicillin allergy after 16 years and 5 standard erysipelas treatments by intravenous benzylpenicillin and/or oral phenoxymethylpenicillin without any symptoms of allergy. It is recommended to analyze specific IgE antibodies for phenoxymethylpenicillin, benzylpenicillin, amoxicillin, and cefaclor to select an appropriate antibiotic. Total number of assays and cases with elevated specific IgE to an antibiotic. The history for dermatitis, allergies, and atopy for the patient, 6 siblings, and parents was negative. The patient had a smoking history of 15 cigarettes a day for 37 years. The medication included only inhaled aerosol, Seretide 50 µg/250 µg .In 1983 and 1996, varicose vein surgeries were performed on the right leg. Seven years later, the patient was treated for an infected right leg wound by using oral cefadroxil.The patient suffered twice from right leg erysipelas in 1990s, and on both occasions, it was treated with oral phenoxymethylpenicillin. In April 2006, the erysipelas was treated first with intravenous (i.v.) benzylpenicillin, then with i.v. cefuroxime followed by oral clindamycin. A relapse occurred 3 weeks later, and it was treated with i.v. and oral clindamycin. Erysipelas reappeared in October 2006, and it was treated, again, with clindamycin. As the prophylaxis of erysipelas, an oral phenoxymethylpenicillin was started but was soon discontinued because of adverse effects in stomach.In October 2007, a surgical operation was performed due to varicose vein relapse and ulcer in the right leg, and the patient received one 3‐gram dose of i.v. cefuroxime.In May 2008, the patient, suffering from the 5th erysipelas, was treated with i.v. clindamycin for 1 day followed by i.v. benzylpenicillin for 3 days. Thereafter, the patient was discharged from the hospital with oral phenoxymethylpenicillin for 3 weeks.9/L. Therefore, the treatment with i.v. clindamycin was continued but without benzylpenicillin. The leg edema and erythema disappeared during the following days, like decreased steadily, also the CRP level and leukocyte count down to 16 mg/L and 10.1 × 109/L, respectively. Upon discharge, oral clindamycin capsules were prescribed for 4.5 weeks without adverse effects.The 6th and severe right leg erysipelas took place 3 months later in early September 2008. In the emergency care unit, the patient was suspected to have an initial sepsis without findings suggestive for necrotizing fasciitis. The patient was planned to be treated with i.v. clindamycin (600 mg × 4 per day) and benzylpenicillin (4 million IU × 4 per day). However, soon after initiation of the drug infusion, that is, within about 30 min, the face became symmetrically swollen, which was considered to be an allergic reaction. The blood pressure decreased down to 96/48 mmHg, the patient received oral cetirizine (10 mg) and i.v. hydrocortisone sodium succinate (125 mg). In the following morning, the patient felt better without fever, and the blood pressure was normalized (124/67 mmHg). Slight facial redness and eyelid edema were seen. However, there was still clear edema and erythema in the right leg. In laboratory analyses, the level of serum C‐reactive protein (CRP) was 198 mg/L and blood leukocyte count was 20.2 × 10In the follow‐up, the patient was free of symptoms, until the 7th right leg erysipelas occurred in August 2014. The infection was treated successfully with i.v. and oral clindamycin.In order to investigate the cause of allergic/anaphylactic reaction during the infusion of clindamycin and benzylpenicillin, laboratory analyses were performed. One week after the allergic/anaphylactic reaction serum total IgE level was markedly elevated, as well as serum levels of specific IgE to benzylpenicillin and phenoxymethylpenicillin. Instead, specific IgE to amoxicillin was normal Table .9/L, ref. 0.1–0.4 × 109/L). Later the patient's eosinophil counts were measured 6 times being within normal range.One month later, serum total IgE was decreased by about 10%. By using a dilution method, a more accurate estimation for the specific IgE was obtained: for benzylpenicillin it was 137.5 kU/L, and for phenoxymethylpenicillin very high 307.5 kU/l. Specific IgE to cefaclor was also elevated, 11.1 kU/L. Blood eosinophilic leukocytes were marginally elevated  and phenoxymethylpenicillin (2.93 kU/L) were markedly decreased. Specific IgE antibodies to amoxicillin and cefaclor were normal.In order to obtain information for comparison and on the prevalence of elevated specific IgE to these antibiotics, a search for laboratory values in the hospital district of Kuopio University Hospital  between January 2010 and August 2021 was performed. The results of 13 other patients are shown in Table Only two patients had elevated specific IgE levels simultaneously to both phenoxymethylpenicillin and amoxicillin. There was one patient with either of these and two patients with simultaneous specific IgE to phenoxymethylpenicillin and cefaclor. One patient showed markedly elevated specific IgE to amoxicillin (11.2 kU/L) without any increase in IgE to phenoxymethylpenicillin and benzylpenicillin.2According to the literature, an anaphylactic reaction to penicillin is very rare, about 0.001% in parenteral and 0.0005% in tablet dosing.The allergic/anaphylactic reaction in the present case was very likely due to i.v. infusion of benzylpenicillin, because of the very high level of specific IgE to benzylpenicillin (137.5 kU/L) and the reaction subsided when continuing with clindamycin alone. Interestingly, this patient had been treated with the same i.v. benzylpenicillin followed by oral phenoxymethylpenicillin about 2–3 months earlier without any adverse effects. Therefore, it is assumed that the patient was sensitized at the end of the oral treatment with phenoxymethylpenicillin. It is, however, possible that the septic‐like erysipelas may have had some additional effect on the clinical outcome. Of note is the observation, that the sensitization with severe clinical reaction can take place unexpectedly even after a heavy and long background of penicillin use for erysipelas.In 2008, serum tryptase analysis was not available in this hospital. This assay might have given additional information about mast cell activation. A positive skin prick‐test and slightly elevated specific IgE levels are not reliable to predict IgE‐dependent penicillin allergy.An interesting finding was that the specific IgE to amoxicillin was normal, even though specific IgE levels to benzylpenicillin and phenoxymethylpenicillin were very high. In the case of amoxicillin, a direct oral exposition can be performed if a mild delayed‐type reaction has occurred. In the literature, there are conflicting views on the direct exposition with amoxicillin, if a patient has experienced an immediate‐type reaction. Therefore, it is not recommended to be used, if an IgE‐mediated reaction has taken place from another penicillin type. However, a direct amoxicillin exposition is considered to be safe, if more than 10 years have gone since the reaction.In the present case, almost 12 years later, serum total IgE level was decreased by about 84%. This is in accordance with previously published data: IgE‐mediated reactions to beta‐lactams have decreased by 80% in 10 years as determined by skin prick tests.The levels of specific IgE to cefaclor were elevated in this case, as also to benzylpenicillin and phenoxymethylpenicillin. The patient had been treated 5 years earlier with an oral 1st generation cephalosporin, cefadroxil. Also, the patient had been treated about 2 years earlier with some i.v. doses and almost one year earlier with a single i.v. dose of 2nd generation drug, that is, cefuroxime, before surgical operation. Cefaclor is, like cefuroxime, a 2nd generation cephalosporin. Since the cross‐reaction between cefuroxime and penicillin is very low,Although the levels of specific IgE to both penicillins and cefaclor in our patient have markedly decreased or even disappeared, it is recommended to avoid beta‐lactam antibiotics and also 2nd generation cephalosporins—until exposition has been performed.Amoxicillin can cross‐react, for example, with cefaclor, cefadroxil, and cefalexin. The ratio between cefadroxil and amoxicillin is 12%, but higher percentages of even 27% has also been reportedThe cross‐reactions of beta‐lactam antibiotics can result from several structures in the molecule: thiazolidine ring (penicillin), dihydrothiazine ring , R1 and R2 side chains, or several positions at the same time, but mostly due to side chains.In the literature, a cross‐reaction between penicillin and cephalosporins has been reported to be at the level of 10%, but thereafter it has been found that the early cephalosporin preparations contained penicillin as contamination, leading to over‐estimation of the cross‐reactivity; the present estimate for this cross‐reactivity is 1%–2.5%.In the clinical practice, physicians may face the situation that penicillin is the most recommended drug. In such a case, desensitization therapy, first with phenoxymethylpenicillin and later with benzylpenicillin, might be possible to perform, but it requires several weeks to complete the procedure.In this report, only 2 men and 12 women, tested positive for elevated specific IgE to an antibiotic, were found in about 12 years. The female predominance over males is in concordance with a recent review that a drug allergy is more frequently reported in adult females than males, and 57.9%–73.9% of the drug‐induced anaphylaxis cases were among females.The search for elevated specific IgE to antibiotics in this hospital district revealed only 5 relevant positive results for penicillin in over 11 years, being in accordance with the estimation elsewhere. However, some rare patients can unexpectedly develop a severe allergy even after a long history of penicillin use, like did the present patient. Also, although he had extremely high levels of IgE antibodies against phenoxymethylpenicillin, benzylpenicillin, and cefaclor, no IgE antibodies were detected against amoxicillin. Therefore, every time an immediate reaction to beta‐lactams develops, it is recommended to analyze specific IgE to penicillins, amoxicillin, and cephalosporin, as well as total IgE, in order to obtain more accurate estimations for allergy and cross‐reactions. This wide spectrum of the allergy potential of penicillin variants and cephalosporins will be of importance for example in oral surgery of bone grafts that an adequate antibiotic is available to reduce the risk of complications.Authors do not have any relevant conflict of interests.Both authors have equally participated in the preparation of this article.Kuopio University Hospital with Ethics Committee has given permit to publish this patient case and the background allergy laboratory data as reference.The patient has given a written consent to publish this patient case.


Immunoglobulin E (IgE) is thought to have evolved to protect mammalian hosts against parasitic infections or toxins and plays a central role in the pathogenesis, diagnosis, and therapy of IgE-mediated allergy. Despite the prominence of IgE responses in most parasitic infections, and in stark contrast to its use in the diagnosis of allergy, this isotype is almost completely unexploited for parasite diagnosis. Here, we discuss the perceived or real limitations of IgE-based diagnosis in parasitology and suggest that the recent creation of a new generation of very sensitive cellular IgE-based reporters may represent a powerful new diagnostic platform, but needs to be based on a very careful choice of diagnostic allergens. IgE is an immunoglobulin isotype only found in mammals  which are the target of a specific IgE response, i.e., which possess one or more epitopes recognized by IgE. ‘Allergenicity’ here is understood as the ability of multivalent allergens to induce activation of cells bearing the high-affinity IgE receptor FcεRI . The intrinsic ability of an allergen to induce a Th2-biased immune response in a naïve host, leading to the synthesis of specific IgE, will not be addressed here. The important property of allergenicity here is the ability of an allergen to engage more than one FcεRI-bound IgE molecule, thereby clustering the receptor and inducing signal transduction, leading to cellular activation and mediator release. In this sense, only molecules that have more than one accessible epitope, within a certain distance of each other, are considered allergenic.Necator americanus, we were able to detect sensitization of peripheral blood basophils as soon as 5–6 weeks after primary infection by performing basophil activation tests . With the caveat of the low number of experimental replicates, this would suggest that anti-parasite IgE responses can become apparent as early as 4 weeks after the original infection, which also matches the earliest time point of basophil conversion found in our work with N. americanus  infection, the absence of detectable IgE levels in serum in the presence of basophil sensitization points to another relatively simple, but currently underexploited fact: That a cellular readout for parasite-specific IgE is possibly superior to traditional methods (such as ELISA) in terms of sensitivity. As will be explained below, IgE crosslinking by matching allergens induces a powerful and fast multi-tiered cellular signal transduction cascade, in which a relatively modest engagement of a small percentage of IgE receptors on the surface results in a full cellular response within minutes after activation  demonstrated very high specificity (99.3%) and sensitivity (100%), although tested serum numbers were relatively small  allergens. In the case of scabies, the occurrence of cross-reactive antibodies against house dust mites as confounding factor has been highlighted , and to a much larger extent also as a source of food allergens  (Homann et al. Schistosoma mansoni in several studies by Michael J. Doenhoff and his co-workers (El-Faham et al. Another important potential source of false-positive results in conventional serological methods caused by CCDs is the occurrence of a high level of cross-reactivity between parasite- and plant-derived glycans recognized by IgG, as shown for With the exception of α-Gal-carrying allergens, the inability of most CCDs to crosslink receptor-bound IgE means that such allergens are unlikely to cause false-positive results in IgE reporter cell line assays, in contrast to assays measuring binding of specific IgE to allergens immobilized on a solid phase (Foetisch et al. Taken together, parasite-specific IgE, despite its ubiquity in the immune response to parasites, is currently under-used as a diagnostic tool, in contrast to the use of allergen-specific IgE in allergic diseases, where it plays a central role. The development of highly sensitive IgE reporter systems may play an important role in overcoming some of the real or perceived issues limiting the diagnostic use of IgE in parasite infections. However, implementing an IgE-based diagnostic system will require a careful choice of suitable allergens. Only then can the performance of such diagnostic systems be tested in a variety of parasitic disease settings.


In this study, Zhao et al. set out to characterize how plants respond to cold through regulation of FLC expression. Using genetics and genomics approaches, the authors reveal how natural temperature fluctuations promote COOLAIR regulation of FLC, with the first autumn frost acting as a key indicator of autumn/winter arrival. Arabidopsis, which involves a multiphase cold-dependent silencing of the floral repressor locus FLOWERING LOCUS C (FLC). Cold-induced transcriptional silencing precedes a low probability PRC2 epigenetic switching mechanism. The epigenetic switch requires the absence of warm temperatures as well as long-term cold exposure. However, the natural temperature inputs into the earlier transcriptional silencing phase are less well understood. Here, through investigation of Arabidopsis accessions in natural and climatically distinct field sites, we show that the first seasonal frost strongly induces expression of COOLAIR, the antisense transcripts at FLC. Chamber experiments delivering a constant mean temperature with different fluctuations showed the freezing induction of COOLAIR correlates with stronger repression of FLC mRNA. Identification of a mutant that ectopically activates COOLAIR revealed how COOLAIR up-regulation can directly reduce FLC expression. Consistent with this, transgenes designed to knockout COOLAIR perturbed the early phase of FLC silencing. However, all transgenes designed to remove COOLAIR resulted in increased production of novel convergent FLC antisense transcripts. Our study reveals how natural temperature fluctuations promote COOLAIR regulation of FLC, with the first autumn frost acting as a key indicator of autumn/winter arrival.Plants monitor many aspects of their fluctuating environments to help align their development with seasons. Molecular understanding of how noisy temperature cues are registered has emerged from dissection of vernalization in  Arabidopsis, a major determinant of seasonal flowering is the floral repressor FLC, which is epigenetically silenced by prolonged cold during vernalization , as well as an extra SV accession, Löv-1, collected from the North Swedish field site   . Recent ol FRI”) .COOLAIR induction in field conditions to determine whether COOLAIR plays a role in natural conditions in different Arabidopsis accessions and their associated NILs. Three climatically different field sites were chosen: Norwich, United Kingdom ; Ullstorp, Sweden ; and Ramsta, Sweden  . In contrast, Col-0  had very low levels of total COOLAIR in Norwich and at most time points in Sweden. COOLAIR levels in the RV accession Edi-0 and its NIL were very similar to Col FRI in Norwich, but in Sweden, especially North Sweden, COOLAIR was higher in Edi-0 than Col FRI. This was also the case for Var2-6, with early differences only in Sweden. These data suggest COOLAIR induction in the different accessions shows different temperature sensitivity.Three SV accessions  showed higher COOLAIR in all genotypes, even Col-0, in the second week of measurements (the 26th day after sowing) in the first North Sweden planting and the sixth week (the 59th day after sowing) in the South . These unusual peaks occurred when temperatures dipped below 0°C on the morning of the day of measurement .We noticed a strong peak of expression of COOLAIR by freezing by reproducing the temperature profile of the week before the first peak in South Sweden in a growth cabinet. COOLAIR expression rose within an hour of experiencing freezing and peaked ∼8 h after freezing but returned to cool-temperature levels within 24 h . There was a small reduction in the level of FLC transcripts immediately after freezing exposure , reflecting the coordination of antisense–sense transcriptional circuitry. To further analyze the interrelationship of freezing, COOLAIR expression, and FLC expression, we grew seedlings for 2 wk in matched chambers at an average temperature of 5°C, but given either as a constant temperature or two different fluctuating temperature regimes repeated every 24 h . Of note, this hyperinduction of COOLAIR is not just a consequence of a steep temperature drop, as COOLAIR induction was lower after transfer from nonvernalized (NV) 20°C to constant 5°C . All the temperature regimes led to a reduction in FLC expression, including the FLC mRNA and the unspliced transcript level (nascent transcript containing intron 2 and intron 3) used as a proxy for transcription  and distal (class II) polyadenylated forms, showed ectopic up-regulation in ntl8-D3 . Two separate mutant alleles, ntl8-D1 and ntl8-D2, and three overexpression transgenics, ntl8-OE1, ntl8-OE2, and ntl8-OE3, showed the same COOLAIR up-regulation  on the ntl8-D3 mutant in warm conditions . The TEX1.0 line was generated in a previous study  (TEX1.0 and TEX2.0 lines (FLC transcript levels in both TEX1.0 and TEX2.0 reduced more slowly than in wild-type controls (transgenic FLC-WT and nontransgenic Col FRI) during vernalization   were gre.0 lines B. In natlization C, supporn TEX1.0 D, likelyn TEX1.0 . Consistnditions E–G, reinCOOLAIR induction led to significantly lower FLC mRNA levels . We found that FLC down-regulation in ntl8-D3 and ntl8-OE3 is substantially suppressed by the COOLAIR knockout . Therefore, COOLAIR up-regulation is a major causal factor for the FLC down-regulation in the ntl8-D or ntl8-OE mutants, supporting a direct role of COOLAIR or COOLAIR transcription in FLC transcriptional shutdown.Given that the freezing-enhanced A levels E, these  regimes H, and FLly lower I. In con reduced H,I. ThisΔCOOLAIR . In bothTEX lines, it became clear that distally polyadenylated COOLAIR was still produced  was performed . In wild-type plants, grown without (NV) or with 2 wk of cold treatment (2WV), two major spliced distal COOLAIR forms with a range of transcriptional start sites were identified . In the TEX1.0 line, antisense transcription was found to initiate within the RBCS terminator fragment, producing a distal COOLAIR with the same splice sites as on the endogenous locus . In the TEX 2.0 line, no COOLAIR transcripts were detected except for the CAS transcript initiating within FLC exon 1 . The multiple transcriptional start sites of CAS (TEX2.0 (COOLAIR (class I) present at very low levels in both TEX lines is cold induced . Thus, the major COOLAIR-CAS transcripts in the TEX lines are novel CAS and are not cold induced  that had been designed to remove COOLAIR from FLC . In addition, similar to TEX1.0, antisense transcript start sites were detected in the MAF2-T terminator in the FLC + MAF2-T line . Similar findings of cryptic promoter usage after disruption of upstream promoters, and alternative transcripts arising when antisense transcription is perturbed, have been reported in S. cerevisiae , suggests a tight interconnection between antisense transcription and chromatin state at the locus.We further investigated three other transgenic lines , likely due to the epigenetic silencing of the whole locus by the VIN3-dependent Polycomb switching mechanism that occurs after the cold-induced transcriptional silencing , GFP-NTL8-D2 transgenic line, and NTL8 overexpression lines  were previously described  for the forward genetic screen.The escribed , into CoFLC::FLC-TEX2.0 was generated by inserting a NOS terminator fragment within the first exon of COOLAIR within a ∼12-kb genomic fragment, using adjacent EcoRI and SalI restriction sites immediately downstream from the FLC sense 3′ UTR and the primers “fragment-1-F-1653” (5′-GCTTAACGAGCTTGCACACA-3′) with “fragment-1-R-EcoRI-1653” (5′-GAGGAATTcaagatctcgatgcaattctcac-3′) and “fragment-2-F-SalI” (5′-AGAGTCGACagtgtatgtgttcttcacttctgtcaa-3′) with “fragment-2-R” (5′-TATGGAAGAGGTCGGTCACG-3′). The assembled fragment was cloned into pCambia1300, which was transformed into the Arabidopsis flc-2 FRI genotype with a floral dipping method. Transformants were selected on medium supplemented with hygromycin (Sigma-Aldrich H0654), and single-copy transformants were selfed to generate homozygous T3 lines. Both FLC and COOLAIR expressions were screened before vernalization and after a 2-wk vernalization treatment to identify a single representative line, TEX2.0-472.Plant were generally grown in growth conditions as described previously .Details for field experiments have been described previously  agar plates without glucose. For microscopy, plants were grown on MS plates with 1% agar placed vertically. For nonvernalized (NV) conditions, plants were grown for 10–12 d at long photoperiod conditions , while for vernalization treatment, plants were moved to 5°C cold treatment at short photoperiod conditions  after growing at long photoperiod conditions for 7 d if not specified.Seeds were sown on plates with selective antibiotic and MS agar media without glucose. Plants were grown for 10 d postgermination in long days at 20°C and then grown for an additional 3 d before NV sampling or moved to a growth room for vernalization with 8-h light and grown for 2 or 4 wk (2WV or 4WV) at 5°C, before transferring seedlings to soil and growing for 20 d at 22°C/20°C with 16-h light/8-h darkness (4WT20). Three replicates of >15 seedlings  or three replicates of three pooled leaf/meristem tissue (4WT20) were screened for expression.TEX1.0 and nothing for TEX2.0.Flowering time was analyzed as previously described . BrieflySupplemental Table S2. Three biological replicates of more than five seedlings were sampled at 17:00 and 20:00 on the seventh day, the night before freezing; at 07:00, 09:00, 11:00, 13:00, 15:00, 17:00, and 20:00 on the eighth day, when freezing occurred at 10:00 and 13:00–14:00; and at 20:00 on the following day.Plants were grown in a nonvernalizing growth chamber in long days for 1 wk at 22°C/20°C 16-h light/8-h darkness and then transferred to a growth chamber (Conviron) with the temperatures and light period matching the week before the first frost  and the day following the morning frost. Temperatures and light times are shown in FRI seeds were sown onto soil. Nonvernalized plants were grown for 10 d in the nonvernalizing growth chamber and sampled at six time points over 24 h before plants were then moved to 8-h light/16-h darkness growth chambers for vernalization Light was on from 09:00 to 17:00. For vernalization, plants were treated with three different temperature regimes: constant 5°C, daily fluctuating 5°C (3°C–9°C) and daily fluctuating 5°C (−1°C to 12°C) .Col to 12°C) B. Three VIN3 expression in warm conditions , similar to what we found in ntl8-D1 and ntl8-D2 mutants .Mutagenesis was carried out following the procedures previously described . Around  mutants . FurtherSupplemental Tables S3 and S4. For field experiments, RNA extraction and QPCR were performed as described (At5g25760 (“PP2A”) and At1g13320 (“UBC”) control genes. The same analysis was also used in Supplemental Figure S3, C–E. The rest results were normalized to single reference gene, UBC. Primers used are described in Supplemental Table S3.Total RNA was extracted as previously described . Genomicescribed , 2020. Intl8-D3 mutant.For detecting the fluorescence of FLC-Venus, confocal imaging was performed using a 20×/0.7 NA multi-immersion lens, with water as the immersion fluid on a Leica TCS SP8 X confocal microscope following the procedures in The smRNA FISH experiment was performed following the procedures described previously .2, 0.5% Triton X-100, 0.5 mM PMSF, proteinase inhibitor cocktail [Roche 04693159001], 5 mM DTT). After nuclei extraction, purified nuclei were lysed by RIPA buffer  and then fragmented by sonication (Diagenode Bioruptor). After sonication, the fragmented chromatin extract was cleared by centrifugation at 13,000 rpm for 15 min at 4°C before immunoprecipitation. The immunoprecipitations were performed with GFP-trap beads (Chromotek GTMA-20) for GFP-NTL8-D2  in the ntl8-OE3 line. Relevant primers are listed in Supplemental Table S3.NTL8 protein ChIP experiments were carried out following the methods described in -NTL8-D2 G and antCOOLAIR-specific primer, RT-R, with TUB6 as a control . At least 48 colonies from each RNA sample were examined by PCR before at least two colonies from each different sized PCR product were sent to Sanger sequencing. Oligos for 5′ RACE are listed in Supplemental Table S4.Five micrograms of total RNA was first treated with calf intestine alkaline phosphatase  for 1 h at 37°C and purified with phenol:chloroform. After 5′ cap removing for 1 h at 37°C with cap-clip acid pyrophosphatase , 1 μL of 5′ RACE adapter 0.3 μg/μL; (5′-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3′) was ligated to a half volume of the CIAP/CCAP-treated RNA (2.5 μL) by T4 RNA ligase (NEB M0204S) for 2 h at 37°C. The CIAP-treated RNA without following CCAP treatment was used as control. All the 5′ RACE adapter-ligated RNA was then reverse transcribed by SuperScript III reverse transcriptase (ThermoFisher 18080093) with a distal  control B. The cD


The phosphoinositide-3-kinase (PI3K)/AKT pathway regulates cell survival and is over-activated in most human cancers, including ovarian cancer. Following growth factor stimulation, AKT1 is activated by phosphorylation at T308 and S473. Disruption of the AKT1 signaling pathway is sufficient to inhibit the epithelial-mesenchymal transition in epithelial ovarian cancer (EOC) cells. In metastatic disease, adherent EOC cells transition to a dormant spheroid state, characterized previously by low S473 phosphorylation in AKT1. We confirmed this finding and observed that T308 phosphorylation was yet further reduced in EOC spheroids and that the transition from adherent to spheroid growth is accompanied by significantly increased levels of let-7 miRNAs. We then used mechanistic studies to investigate the impact of let-7 miRNAs on AKT1 phosphorylation status and activity in cells. In growth factor-stimulated HEK 293T cells supplemented with let-7a, we found increased phosphorylation of AKT1 at T308, decreased phosphorylation at S473, and enhanced downstream AKT1 substrate GSK-3β phosphorylation. Let-7b and let-7g also deregulated AKT signaling by rendering AKT1 insensitive to growth factor simulation. We uncovered let-7a-dependent deregulation of PI3K pathway components, including PI3KC2A, PDK1, and RICTOR, that govern AKT1 phosphorylation and activity. Together, our data show a new role for miRNAs in regulating AKT signaling. Epithelial ovarian cancer (EOC) has a 5-year relative survival rate of only 44%, making it the fifth most lethal cancer among women, largely attributed to high rates of metastasis. EOC metastasis begins when tumor cells are shed into the peritoneal cavity where they exist in suspension as single cells or multicellular aggregates known as spheroids. As spheroids, EOC cells survive in a dormant state, evading apoptosis and enabling metastasis. EOC cells utilize the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway to dynamically regulate the dormant-to-proliferative metastatic switch. The PI3K/AKT/mTOR pathway controls the phosphorylation and thus activation of the three AKT isoforms , AKT1, AKT2, and AKT3 . SurprisAKT1 activity is regulated by phosphorylation at two key sites, T308 and S473 . The phoMultiple recent studies are establishing new connections between miRNAs and AKT1 signaling. AKT1 is a target of different miRNAs that regulate AKT1 protein levels in cancer cells. Binding of miRNA-422a to the 3′-untranslated region (UTR) of AKT1 leads to decreased production of AKT1, resulting in reduced tumor growth and proliferation in colorectal cancer cells. Conversely, down-regulation of miR-422a accelerates tumor growth by releasing its regulation of the PI3K/AKT pathway . StudiesMiRNAs and their regulatory pathways have emerged as new therapeutic targets for drug-resistant cancers. The conserved miRNA let-7a is a known regulator of oncogenesis that suppresses tumor proliferative activities by repressing oncogenic signaling pathways. Verified targets of let-7a include the oncogenes c-Myc, Rat sarcoma virus (Ras), high-mobility group A (HMGA), Janus protein tyrosine kinase (JAK), signal transducer and activator of transcription 3 (STAT3), and novel Np95/ICBP90-like RING finger protein (NIRF) . Let-7a Motivated by the above studies that established the ability of miRNAs to regulate the AKT1 pathway as well as those documenting the association of let-7a with tumor cells, we hypothesized that let-7 family miRNAs may act to deregulate PI3K/AKT signaling. Thus, we investigated the impact of miRNAs from the let-7 family on AKT1 phosphorylation status and activity. We identified an inverse relationship between let-7a levels and AKT activity in adherent and spheroid EOC cells that we used as a well-established model of EOC metastasis. We also found AKT1 signaling was deregulated in EGF-stimulated cells supplemented with let-7 miRNAs, which were characterized by enhanced AKT1 signaling, increased phosphorylation at T308, and decreased phosphorylation at S473 of AKT1. Finally, we showed that let-7a deregulates the PI3K pathways components PIK3C2A and RICTOR to control AKT1 phosphorylation status. These data are the first report of let-7a-dependent regulation of AKT1 phosphorylation at both key regulatory sites.2 chamber. TOV-21G plates at 70–90% confluency were split into either standard tissue culture-treated plastic (adherent) or ultra-low attachment  plates at the same dilution. Cells were grown on standard or ULA plates for 3 days before harvesting. Spheroid cultures were centrifuged to pellet cells and washed once with HBSS before trypsinization. Adherent cultures were harvested by standard trypsinization procedures. All washes and incubations were maintained for the same period between adherent and spheroid cultures, including trypsin treatment. Cell pellets washed with PBS were stored in −80 °C until RNA or protein extraction.HEK 293T cells and COS-7 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. TOV-21G cells were grown in DMEM:F12 supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were grown at 37 °C in a 5% COSixty femtomoles of miRNA let-7a/b/g  was usedTotal RNA was extracted from mammalian samples using miRNAeasy Mini Kit (Qiagen). Primers for reverse transcription and quantitative PCR are listed in 2HPO4, 1 mM Na4P2O7, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, and 1% Triton X-100 supplemented with 300 µM PMSF, 1 mM dithiothreitol, and protease inhibitor cocktail (Roche)) and incubated on ice for ~10 min with vortexing. Lysates were clarified by centrifugation at 14,800× g for 10 min at 4 °C. Protein concentration was measured by Bradford, and equal amounts were loaded for each sample. Cell pellets were resuspended in ice-cold lysis buffer 1 (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, and 0.1% sodium dodecyl sulfate (SDS) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF)) or ice-cold lysis buffer 2 , and data were compiled and normalized in Microsoft Excel. RT-qPCR data were analyzed by the ΔΔCt method in Microsoft Excel. EOC cells are a well-studied model system for AKT1-driven cancers. The dormant-to-proliferative metastatic switch in EOC cells is directed by changes in AKT1 phosphorylation status ,3. CellsWe tested the AKT abundance and phosphorylation status A,B in boThe let-7 miRNA family is a well-known suppressor of cancer and metastasis , promptiAs previous literature shows multiple miRNAs impacting AKT protein abundance ,13,14, wAs the phospho-specific antibodies for AKT sites T308 and S473 do not sufficiently distinguish between the three AKT isoforms, we decided to use an established mCherry-AKT1 overexpression construct , allowinIn HEK 293T cells, as expected , EGF stiSince our data show that let-7a increases T308 phosphorylation but decreases S473 phosphorylation of AKT1 in stimulated cells, we investigated the overall impact on AKT1 activity by examining the downstream phosphorylation of a known AKT1 target. We measured the phosphorylation status of the AKT1 substrate glycogen synthase kinase-3 (GSK)  in HEK 2Similar to HEK 293T cells, COS-7 cells stimulated with EGF and transfected with let-7a showed the same significant and specific increase in relative GSK-3β phosphorylation E,F, indiIn addition to let-7a, we measured the expression of the let-7 miRNA family in TOV21G EOC cells and found upregulation of let-7b in spheroids relative to adherent cells, though to a lesser extent than let-7a, while let-7g was unchanged E. To assS473 when control cells are EGF stimulated , which recruits phosphoinositide 3-kinases (PI3Ks) to the membrane. PI3Ks in turn phosphorylates the phosphatidylinositol 4,5-bisphosphate  P2; PIP2) to phosphatidylinositol -trisphosphate P3; PIP3), which activates AKT1 by recruiting both PDK1 and AKT1 to the membrane, leading to phosphorylation at T308 . In concPIK3C2A  in response to let-7a transfection. We observed a significant 1.5-fold increase in PIK3C2A protein levels in cells supplemented with let-7a B,C. No dPIK3C2A D, indicaPIK3C2A  and downPIK3C2A .AKT1 S473 is phosphorylated by the mammalian target of rapamycin (mTOR) complex 2, (mTORC2) , a sevenThe over-activation of the PI3K/AKT signal transduction pathway in response to extracellular signals is a crucial step leading to the progression of cancer and tumorigenesis ,32. WhilT308 levels of all isoforms in spheroids compared to adherent cells. We found TOV2-1G spheroids have reduced phosphorylation at T308 and reduced S473 is a function of reduced total AKT protein levels. This is the first report of reduced T308 phosphorylation in EOC spheroids.The shift from adherent to spheroid cells during EOC metastasis has previously been reported to be AKT-dependent ,38,39,40KRAS mutant cancer cell lines, where high let-7b alone had no effect but lead to decreased phospho-AKT levels when combined with chemotherapies [KRAS mutation. The combination of the findings we report here and previous studies investigating let-7 miRNAs in cancer environments indicate a broad role for this family in cancer progression and metastasis.Several let-7 family miRNAs change expression during cancer metastasis (reviewed in ), implicherapies . The TOVWe here set out to investigate the negative correlation between let-7 abundance and AKT abundance, phosphorylation, and activity that we observed in EOC cells. In independent experiments, we transfected these three let-7 isoforms into HEK 293T cells. In HEK 293T cells, let-7a transfection alone does not alter the abundance of any of the three AKT isoforms. Although no let-7a binding sites are found in the AKT1 3′UTR, the 3′UTR of AKT2 mRNA encodes one potential let-7a binding site, and the 3′ UTR of AKT3 mRNA encodes three potential let-7a binding sites ,27. A prThe S473 residue is located in the AKT1 hydrophobic motif, a regulatory loop distant from the active site ,47. In aS473 levels in gastric cancer cells, where let-7a negatively regulates translation of RICTOR mRNA [S473 levels similar to control cells but are unable to respond to EGF stimulation due to reduced RICTOR levels.AKT1 S473 is phosphorylated by the mTORC2 complex . The mecTOR mRNA . Our datT308 retains most of the activity of the doubly phosphorylated ppAKT1T308,S473 in phosphorylating GSK-3β [Since S473 phosphorylation modulates AKT substrate selectivity ,36, the g GSK-3β . The obsg GSK-3β . Future T308 is located centrally to the AKT1 peptide binding and active sites. Phosphorylation at T308 is necessary and sufficient to activate AKT1 and provide maximal AKT signaling in COS-7 cells  and chicWe showed that the increased phosphorylation of T308 in stimulated cells supplemented with let-7a results from an increased level of the kinases PDK1 and PIK3C2A that are upstream of AKT1. PIK3C2A is a class II PI3 kinase that is activated in response to EGF and insulin stimulation , transduIn addition to increased PIK3C2A levels, we also observed an increase in the protein level of the AKT1 T308 kinase, PDK1. PDK1 can be positively or negatively regulated by its interaction with several proteins, such as collagen type XI aplha1 (COL11A1), short-form Ron (sfRON), Metadherin (MTDH), tongue cancer resistance-related protein-1 (TCRP1), and the tumor suppressor candidate 4 (TUSC4) , yet fewIn summary, we observed that high let-7a levels result in an increased abundance of PIK3C2A and PDK1, which magnifies the ability of cells to respond to EGF stimulation, leading to hyperphosphorylated and hyperactive AKT1. We presented the first evidence of a pathway linking miRNA let-7a levels with AKT1 phosphorylation status, where let-7a also controls the PI3K/AKT cell survival pathway. Several miRNAs are well known to regulate AKT1 abundance, and AKT1, in turn, regulates miRNA abundance via the terminal nucleotidyltransferase TENT2 . We previously showed that AKT1 negatively regulates TENT2, which in turn regulates let-7a stability ,61. The Mycobacterium tuberculosis infection by targeting A20, a feedback inhibitor of the NF-κB pathway [Interestingly, different let-7 family members fulfill different functions in regulating AKT activity. A previous study showed that insulin-PI3K-mTOR signaling is suppressed by let-7f, decreasing AKT phosphorylation at S473 and T308 when cells were stimulated with insulin . Let-7f  pathway . These dIn summary, we described the first evidence of regulation of AKT1 at both the protein and phosphoprotein levels by let-7 family mRNAs. In epithelial ovarian cancer cells, increased let-7 miRNA abundance was identified in spheroid cells, implicating a new role for the let-7 family in AKT1-dependent metastasis. We found that the miRNA let-7a controls AKT1 phosphorylation by deregulating PI3K pathway components, including PI3KC2A, PDK1, and RICTOR. Indeed, in cells treated with different let-7 miRNAs, we found AKT1 S473 phosphorylation was insensitive to growth factor stimulation. Let-7a uniquely deregulated AKT1 signaling by enabling hyper-phosphorylation of T308 and hyper-activity in AKT signaling. Together, the data reveal the ability of the let-7 family of miRNAs to regulate the PI3K/AKT pathway in human cells.