Silent atrial fibrillation (AF) is common. In some patients, it is the only manifestation of AF, while in others, the AF may be symptomatic or both symptomatic and asymptomatic. Regardless, however, to date, the significance, detection, and management considerations for silent AF have been incompletely elucidated. This current study aimed to review, for both the current clinician and investigator, considerations and attitudes and the ongoing studies, respectively, with respect to silent AF. The methods used were a literature review and personal trial and clinical experience; the frequency of silent AF, concerns regarding silent AF, methods to detect silent AF, and prospective trials focused on the detection and management of silent AF were considered. The results of the literature search indicated that recently conducted relevant trials, such as PREDATE AF, ASSERT-II, and REVEAL AF, have shown that silent AF is frequent in patients with risk markers for AF and stroke in whom no prior AF history is present, and in whom no pacemaker or implantable cardioverter-defibrillator implantations have been previously performed. Furthermore, the GLORIA-AF Registry has reported the observance of more permanent AF and more prior strokes in asymptomatic patients. Ongoing trials such as ARTESiA and NOAH-AFNET 6 are expected to clarify the benefits and risks of oral anticoagulation in patients with silent AF. At present, when silent AF is detected in patients with stroke risk markers, most practitioners initiate an anticoagulation regimen. Wise Words and Wives’ Tales: The Origins, Meanings, and Time-Honored Wisdom of Proverbs and Folk Sayings Olde and New by Stuart Flexner and Doris Flexner , this phrase was first stated in a proverb in the Judaic biblical commentaries called the “Midrash” (ca 600).“If speech is silvern, then silence is golden.” According to 1 they are not limited only to symptomatic AF. Specifically, a requirement of symptoms is not part of the guidelines-stated treatment algorithms regarding anticoagulation. Equally important, the issue of silent AF is one with major consequences, as heart failure, stroke, systemic embolism, and death may be clinical consequences of previously undiagnosed AF.2 Likely, their risk relates not only to the burden of AF, but also to the type and severity of associated conditions. Accordingly, significant morbidity and occasionally mortality may result under the circumstances of undiagnosed AF and the consequent lack of prophylactic treatment interventions proven to reduce AF’s adverse consequences. Early detection of undiagnosed AF in high-risk patients may therefore allow for earlier initiation of lifesaving/life-improving therapy. This manuscript will review the issue of silent AF, its significance, its detection, and its management considerations for the interested clinician and investigator. In it, the term silent AF is used in its broadest sense—that is, cases in which AF, though present, is either unrecognized, or it is known but is asymptomatic and therefore not detected by the patient when it occurs. Finally, one caveat: among silent AF, some might include persistent AF that is not recognized by the patient until he/she realizes following cardioversion that their quality of life has improved, and that they did have some low-level symptoms that had been unappreciated.3 The latter are not the patients this manuscript discusses; nonetheless, the same concerns regarding prophylactic anticoagulation and rate control are equally applicable to them.While we can debate whether it is more often preferable to be verbally vocal or silent, can we say the same about atrial fibrillation (AF)? Although some patients are aware of each and every episode of AF, many are aware of only some, and some are even aware of none. The latter is commonly referred to as “silent AF” . Importantly, though AF guidelines exist for rate and rhythm control, the use of anticoagulation, and for other aspects of optimal clinical care,6 with some reports noting that up to 70% of episodes are silent. Silent AF episodes are commonly shorter and slower than symptomatic episodes in patients who have both, yet paroxysmal silent AF has been shown via the monitoring capabilities of implanted devices to be capable of lasting 48 hours or longer.7 In AFFIRM, 12% of AF patients were asymptomatic at baseline, where, compared with symptomatic patients, silent AF was seen more often in men with a longer duration of AF, a lower maximum heart rate during AF, and better LV function.8 In some cases, silent AF is only recognized after the patient presents with a consequence, such as heart failure or thromboembolism.9 Following catheter ablation for AF, post-procedure monitoring shows that the percentage of recurrent AF that is silent, rather than symptomatic, increases.11 Modeling studies, such as that by Turakhia et al.,12 have estimated a high burden of clinically significant silent AF. The global burden is unknown. Finally, the determined prevalence of silent AF is directly related to the intensity of monitoring: the more prolonged the monitoring, the more silent AF becomes apparent. Furthermore, symptoms alone do not provide an accurate assessment of the presence of AF or of its quantity (burden).13In patients with known AF, silent AF is not uncommon. At least one-third of patients with symptomatic AF are found to also have asymptomatic episodes when they are monitored,14 or stroke in appropriate clinical circumstances , while with regards to silent AF specifically, the onset may be more acute or unanticipated or the incidence higher because unrecognized silent AF is less apt to have resulted in a rate control or oral anticoagulant having been prophylactically prescribed. Moreover, and very importantly, atrial thrombogenesis does not automatically accompany patient awareness of AF. The atrial alterations that can be prothrombotic, including atrial dilation, altered histology, impaired contractile and endothelial function, and stasis, are dependent upon the presence of AF and changes that may be resultant from age and/or comorbidities, but are not dependent upon the patient having palpitations, fatigue, or other AF-related symptoms. Accordingly, symptoms are not part of the risk-scoring systems for stroke, such as CHADS2 or CHA2DS2-VASc, and the 2014 American Heart Association/American College of Cardiology/Heart Rhythm Society Guidelines for the Management of Patients with Atrial Fibrillation specifically state that when balancing the risks and benefits when selecting an antithrombotic regimen, that “AF, whether paroxysmal, persistent, or permanent, and whether symptomatic or silent” should be considered.1 The GLORIA-AF Registry has also reported the occurrence of more permanent AF and twice as many prior strokes in patients with asymptomatic AF than in those with AF-related symptoms.15The concerns regarding silent AF are similar to those associated with symptomatic AF, aside from the addition of impaired quality of life that symptoms from AF may produce. That is, both symptomatic and silent AF may result in heart failure17 Notably, Botto et al.16 demonstrated that the higher the CHADS2 score was, then less AF was required to result in thromboembolic events. The interplay between the AF, any associated disorders, and their combined effect on the left atrium (LA) also provides an explanation for the observation that stroke in patients with AF is not necessarily directly synchronous with the time at which AF is present or terminates. The abnormal atrial milieu that underlies thrombogenesis and embolism may still be present when an episode of AF ends if the abnormalities produced by the comorbidities and by the overlying atrial tachycardiac myopathic changes induced by the AF itself are still present and significant.The risk of thromboembolism with AF appears to be related both to the type and severity of the associated disorders, and to the duration of the periods of AF (the AF burden).27 It is important to recognize, though, that patients with pacemakers or ICDs typically have electrical disorders other than AF, and thus that the reported incidence of silent AF and its epidemiological outcome risks may not be identical in patients without these associated disorders.The fact that silent AF is associated with an increased risk for thromboembolism has perhaps best been demonstrated to date by studies that have been performed involving patients with implanted pacemakers or implantable cardioverter-defibrillators (ICDs), and in studies of patients with cryptogenic stroke.24 In the latter, the stroke risk has been associated with relatively modest amounts of AF such that AF durations of five to six minutes, 3.8 to 5.5 hours, or of 24 hours have been associated with increased hazard ratios for thromboembolism of 2.2 to 9.4.24 Of note, in a subgroup analysis of one of these, the ASSERT study, the stroke risk was significant only with an AF duration of at least 24 hours, but was not significant with either an AF duration of six minutes to six hours, or six hours to 24 hours.28 Importantly, however, the cited trials vary in their threshold criteria for AF, the demographics of their study populations, the duration of follow-up, and other critical characteristics that make them difficult to compare directly. Also, in ASSERT, the CHADS2 score was only 2.2, whereas in patients with a higher risk score a lesser AF burden may be enough to trigger clot formation.17 Nonetheless, taken together, the link between silent AF and stroke seems consistent.Multiple reports exist that document pacemaker/ICD-detected silent AF occurring in patients with no prior AF history; reports that associate silent AF in pacemaker/ICD patients with an increased incidence of stroke also exist.27 While in the latter, it is possible that the AF could just be a bystander event, rather than being causally linked, the causality is clearer when previously unknown AF is present in a patient who is admitted with acute stroke. The causality is also strongly suggested by observations that anticoagulation of patients with monitoring-detected AF post stroke has reduced recurrent stroke in patients in whom prolonged monitoring was performed, as compared with in those who underwent shorter periods of monitoring and in whom AF was detected less frequently.29 The Find-AFrandomized study showed that more prolonged monitoring detected more silent AF, which led to more anticoagulation use, which was associated with a 50% reduction in the rate of recurrent ischemic stroke.29Corroborating evidence relating silent AF and stroke is available from studies conducted involving stroke patients in whom AF was first reported at the time of the stroke, and in patients with cryptogenic stroke in whom long-term monitoring after the stroke demonstrated a substantial incidence of silent AF during follow-up.30 For example, in a review of monitoring reports of 600 patients randomly selected from over 100,000 tracings from a commercial monitoring database , my colleagues and I reported that clinically important arrhythmias were detected by 24-hour Holter monitoring in 6.2%, versus in 36% using 30-day auto-triggered memory loop recording.30 Additionally, in that data set, 24-hour Holter monitoring detected AF (including both symptomatic and silent) in 27 patients, as compared with in 146 patients with 30-day recordings.30 Similarly, the FIND-AF investigators presented data showing that three 10-day Holter recordings in post-stroke patients detected AF in 13.5% by six months, as compared with 4.5% in routinely assessed patients.29 Additionally, the EMBRACE investigators reported AF lasting at least 30 seconds in 16.1% of cryptogenic stroke patients with a 30-day event-triggered recorder versus 3.2% of patients in the control group, while the CRYSTAL AF investigators reported cumulative AF detection rates of 3.7%, 8.9%, 12.4%, and 30.0% at one, six, 12, and 36 months using an implantable loop recorder (ILR) versus a rate of 3.0% at 36 months in their control group.27 Moreover, in monitoring studies assessing AF with ILRs, the median time to first detection has routinely been longer than 30 days. Recently, smartphones have also begun to be utilized to detect AF. However, while they may be useful as alternatives to older patient-applied recorders at the time of symptomatic periods, or as a means to detect long-lasting paroxysms of AF, because of the brief and intermittent nature of their monitoring capabilities they are not optimal options for the detection of silent AF in the overwhelming majority of patients who are at risk, or for adequately evaluating AF burden, and have yields that do not compare with those of the prospective trials discussed below that utilized insertable monitors.31It is abundantly clear from the medical literature that the longer patients with intermittent arrhythmias are monitored, the greater the arrhythmia documentation will be. Thus, ambulatory monitoring is superior to random electrocardiograms (ECGs), and prolonged monitoring is superior to 24 to 48 hours of Holter monitoring.. They are PREDATE AF, ASSERT-II, and REVEAL AF.34 Additionally, a larger but similar in nature trial, LOOP (NCT02036450), is still ongoing, while another small trial, GRAF-AF (NCT01461434), has not yet been presented or published.Notably, device-detected silent AF is not limited to studies in patients with pacemakers/ICDs or post-stroke patients. Recently, three prospectively performed trials have been reported that considered patients with risk markers for AF and for stroke who demonstrated no prior history of AF, incorporating long-term monitoring (in years) with an implanted/inserted cardiac monitor (ICM) 32 was an investigator-initiated, prospective, single-arm, open-label, single-center trial with a planned enrollment quota of 350 asymptomatic subjects with a CHA2DS2-VASc score ≥ 2 and no history of AF who were monitored using ICMs. However, it was stopped prior to enrolling the full 350 subjects, as it met its power analysis goals early. The primary outcome was the new onset of AF ≥ six minutes in duration, as evaluated by a review of monthly ICM transmissions. Secondary outcomes included the time to first detection of AF, the comparison of AF incidence by gender, the comparison of AF incidence in subjects with high versus low CHA2DS2-VASc scores, and the characterization of treatment patterns of patients found to have AF. Ultimately, 249 patients were enrolled, with 245 (98.3%) included in the final study analysis . During the study, three patients died due to non-cardiac causes; their data were included through their last monthly transmission. Additionally, five patients required pacemaker implantation during the course of the trial; these individuals continued in the trial per design. The mean age was 74.3 years, the mean CHA2DS2-VASc score was 4.6, the mean left ventricular ejection fraction was 81%, and 41% of the study subjects were female. The use of an antiarrhythmic drug at the time of enrollment was an exclusion criterion, but the use of β-blockers was allowed and were being taken by 61%. Anticoagulants were not taken at baseline, but 83% of subjects were using aspirin. The most common comorbidity was hypertension (over 95%), and about 20% had a remote stroke history.PREDATE AF2DS2-VASc scores were compared . However, there were significantly more females in the group with a high CHA2DS2-VASc score ; additionally, the female subjects (who are much less likely to develop AF at a given age), had approximately half the AF incidence as that of the men . Following notification that AF was present, caregivers elected to initiate oral anticoagulation (OAC) (which was not protocol driven) in 76.4% of patients, with either a novel oral anticoagulant (n = 38) or warfarin (n = 4). PREDATE AF therefore demonstrated that at least in this one small trial, silent AF appears to be common in patients who do not have a history of AF but in whom high-risk markers for AF are present, and that physicians deem such AF to be important enough to warrant OAC in most of these individuals.During an average follow-up of 451 ± 185 days, a total of 55 patients were diagnosed with AF (n = 54) or atrial flutter (n = 1), resulting in an overall detection rate of 22.4% . In the patients in whom AF was found, 27.3%, 50.9%, 69.1%, and 89.1% were detected in the first, third, sixth, and 12th month, respectively. Fifty-one (92.7%) were asymptomatic; the mean ventricular rate was 112.2 ± 30.3 bpm. There was no significant difference in the AF-free survival when patients with high (5–9) and low (2–4) CHA33 was a similarly sized but multicenter trial with a single-arm, open-label protocol that enrolled 273 patients who did not have a history of AF but who had risk markers for it, and who were given an ICM for AF detection. ASSERT-II enrolled patients aged ≥ 65 years who were being cared for in a cardiology/neurology clinic and who had either a CHA2DS2-VASc score ≥ 2, obstructive sleep apnea, or a body mass index > 30; and either a LA volume ≥ 58 mL or a LA diameter ≥ 4.4 cm, or a serum N-terminal prohormone of brain natriuretic peptide ≥ 290 pg/mL. Two hundred fifty-six patients received the ICM, four died, and 252 completed a minimum of nine and a maximum of 16.3 months of follow-up. Hypertension was present in 73%; additionally, 48% had a prior stroke, transient ischemic attack, or systemic embolism; 9% had a heart failure history; the mean CHA2DS2-VASc score was 4.14; the mean age was 74 years; and 34% were female. The primary endpoint was subclinical AF (SCAF) lasting at least five minutes. The incidence of AF ≥ five minutes was 34.4% per person-year. At 18 months, AF lasting ≥ 30 minutes, ≥ six hours, and ≥ 24 hours was 21.8%, 7.1%, and 2.7%, respectively. Using baseline characteristics, of the subgroups examined, a LA volume of at least 73.5 mL was a predictor of AF (hazard ratio (HR): 1.85; p = 0.015). AF was also more common in older patients . Interestingly, the systolic blood pressure (140 mmHg versus 135 mmHg), history of heart failure (11.4% versus 3.3%), the presence of diabetes (28.9% versus 17.8%), and the presence of vascular disease (36.1% versus 24.4%) were higher in those patients without versus in those with AF detected. For the presence of AF ≥ five minutes, only LA volume remained significantly different. A stroke history was no more common in those with AF detected than in those without. Similar to PREDATE AF, the ASSERT-II investigators concluded that SCAF is common not only in patients with pacemakers, but also more broadly in elderly individuals with specific risk markers.ASSERT-II34 was a prospective, single-arm, open-label, multicenter trial that assessed 446 patients who did not have a history of AF but who had high-risk markers for it. Three hundred ninety-four of these individuals underwent ICM insertion for silent AF, with 385 being eligible for analysis. Fifty-two of the 446 patients were excluded, primarily due to an inability to meet the inclusion/exclusion criteria, or at the patient’s request. Nine of the 394 were further excluded from analysis because of an inclusion criteria violation, use of an antiarrhythmic drug, or no post-insertion data. Inclusion criteria included a CHADS2 score of ≥ 3 or a score of 2, plus at least one of the following: coronary artery disease, renal impairment (glomerular filtration rate: 30 to 60 mL/min), sleep apnea, or chronic obstructive pulmonary disease. At least 70 patients were required for each CHADS2 group . Device transmissions were made monthly and in-office follow-up visits occurred every six months for a minimum of 18 and a maximum of 30 months. All included patients were required to have a minimum of 24 hours of external ECG monitoring within 90 days prior to enrollment or device insertion in which no AF was detected. This inclusion criterion was unique to REVEAL AF and was not used in either PREDATE AF or ASSERT-II. The primary outcome was the incidence of adjudicated AF ≥ six minutes at 18 months. Secondary and additional exploratory objectives included predictors of AF, physician actions (OAC prescription in particular) in response to AF detection, AF incidence at additional time points from 30 days to 30 months, a comparison of AF incidence among the CHADS2 subgroups, and the median time from implant to AF detection.REVEAL AF2 score was 2.9, and the mean CHA2DS2-VASc score was 4.4.In REVEAL AF, the mean age was 71.6 years, hypertension was present in 93.7%, 20.6% had a history of heart failure, 59.1% had coronary artery disease, 62.9% had diabetes, 49% were women, the mean CHADS2 subgroups. The median time to detection was 123 days. Hence, AF would not have been detected in this trial in over three-quarters of the patients had monitoring been limited to one month. Similarly, the rates of AF detected in both PREDATE AF and ASSERT-II were also quite low in the first month. Only age and body mass index were predictors of AF out of all the baseline demographic criteria assessed. Among the patients who met the primary endpoint, 56.3% were prescribed an OAC. As in PREDATE AF, this action was not protocol driven. Thirteen patients died following enrollment, but no deaths were related to the device in any way. Most of the patients had one or more non-specific symptoms at baseline (approximately 10% had none), but there were no differences in outcome events in those with versus those without symptoms.35 This finding is not really surprising, since symptoms have been proven to have a poor correlation with AF in many studies.39The primary endpoint in REVEAL AF occurred in 29.3% at 18 months. Of these 128 subjects, at least one AF episode was ≥ 24 hours in 10.2%. Adjudicated AF of ≥ six minutes was also present in 40.0% at 30 months. It was 6.2% at 30 days. There were no significant differences in these events across the three CHADSThus, REVEAL AF, a somewhat larger and longer trial than PREDATE AF or ASSERT-II, confirmed a significant incidence of previously undiagnosed AF in a group of patients identified demographically to be at-risk for both AF and stroke, in whom a history of AF was absent and no previously implanted pacemaker/ICD was present. The at-risk populations studied in REVEAL AF, PREDATE AF, and ASSERT-II are a common group of patients encountered in clinical practice. The willingness of physicians to have their patients enroll in such trials and their high rate of initiation of OAC therapy when AF is detected reveals their now-evident concern regarding the presence of silent AF. However, the initiation of unblended, open-label anticoagulation in these trials precludes the assessment of the risk of stroke or the certain benefit of anticoagulation in them.2DS2-VASc score of at least 4. NOAH-AFNET 6 is an investigator-initiated, prospective, parallel-group, double-blind, randomized multicenter trial assessing the prevalence of stroke, symbolic embolism, and death in patients aged at least 65 years old who have a CHA2DS2-VASc score of at least 2 and pacemaker/ICD detected silent AF treated with edoxaban versus aspirin at 100 mg/day. All of these trials are expected to be completed between 2019 and 2021.Accordingly, it is unproven whether OAC treatment of silent AF episodes, especially if relatively short, can significantly reduce the risk of thromboembolism. Two trials, ARTESiA (NCT01938248) and NOAH-AFNET 6 (NCT02618577) are underway to assess the potential role of OAC in patients with device-detected AF. ARTESiA is a prospective, randomized, double-blind study designed to determine if treatment with standard-dose apixaban versus aspirin at 81 mg/day will reduce the risk of ischemic stroke and systemic embolism in patients with device-detected silent AF of at least six minutes, but not of > 24 hours, in duration, plus a CHAFigure 2.Finally, we physicians who care for our fellow humans are not the only ones struggling with silent AF. Our veterinarian colleagues do as well. Clinically meaningful silent AF is not just a disorder of humans: it occurs in many species, including in chimpanzees, our genetically closest and now endangered relatives. Importantly, 40% of male chimpanzees in captivity die of cardiac disease—a dilated, fibrotic, and sometimes fat-replaced cardiomyopathy. Some have had rapid AF demonstrated. Whether this is a consequence of the cardiomyopathy or the cause of it has not yet been determined. Certainly the chimps do not complain of symptoms, yet the AF is serious. An example, obtained with an ILR, and hence pertinent to the above discussion, is shown in In conclusion, the final points are to be remembered:Silent AF appears to be relatively common—at least in patients with high-risk factors for AF and stroke, and in patients with electrical disorders that require implantation of a therapeutic electrical device.randomized trial discussed above, plus the current AF guidelines, as well as the physician actions demonstrated in the silent AF trials cited, suggest that the anticipated answer will be yes.Silent AF likely carries a risk of thromboembolism when it occurs in the setting of stroke risk factors, especially if the burden is high and/or the comorbidities are marked. However, the definitive answer as to the benefit of initiating OAC in patients with device-detected silent AF will remain unsettled for the present. Nonetheless, the findings reported in the Find-AF12The less-settled questions include what will be the threshold for AF burden above which risk-based treatment is justifiable, and who or what are the optimal populations to be screened? Because AF burden and comorbidity severity are synergistic, it is unlikely that a single silent AF duration or frequency will be universal; rather, it will likely vary inversely with the type and severity of the concomitant conditions present in each patient. Additional studies are needed to further elucidate the characteristics of silent AF that are clinically meaningful, and what form optimal therapy will take. Presumably, some of this will come out of the recently established AF-SCREEN international collaboration.


Zheliang, and 91.4% with 6.7% for Xiushui, respectively, using the least squares-support vector machine. Moreover, the robustness of the model was validated through transferring the model of Zheliang to Xiushui with the overall accuracy of 90.3% and false negative rate of 7.8%. These results demonstrate the feasibility of the developed system for RFS identification with a low detecting cost.Spectral imaging is a promising technique for detecting the quality of rice seeds. However, the high cost of the system has limited it to more practical applications. The study was aimed to develop a low-cost narrow band multispectral imaging system for detecting rice false smut (RFS) in rice seeds. Two different cultivars of rice seeds were artificially inoculated with RFS. Results have demonstrated that spectral features at 460, 520, 660, 740, 850, and 940 nm were well linked to the RFS. It achieved an overall accuracy of 98.7% with a false negative rate of 3.2% for  Ustilaginoidea virens (U. virens) Takah (teleomorph Villosiclava virens), which can attack the stamen filaments of rice at the booting stage [Digitaria marginata, Imperata cylindrica, and Panicum trypheron are also alternative hosts of U. virens [U. virens detection with high sensitivity and accuracy [Rice false smut (RFS), also called as pseudo-smut, is caused by a pathogenic ascomycete fungus named ng stage ,2. Apart. virens . It can . virens . It has . virens ,6. Conseaccuracy . HoweverDeveloping a rapid and non-destructive technique for the quality evaluation of agricultural plant seeds has attracted a great interest in different research backgrounds such as variety identification in maize seeds ,9; germiTo our knowledge, few studies have been carried out to detect rice false smut (RFS) in rice seed using a low-cost imaging system. Therefore, the main objective of this research was to develop a low-cost narrow band multispectral imaging system and establish a feasible classification model for detecting rice false smut (RFS). To achieve this goal, the specific objectives were to (1) develop a low-cost multispectral imaging system with six narrow bands distributed in the visible/near infrared (Vis/NIR) region; (2) acquire the multispectral images of healthy and RFS infected samples of two different cultivars of rice seeds; and (3) establish a feasible classification model for rice false smut (RFS) discrimination.Zheliang and Xiushui that are susceptible to rice false smut (RFS) were used in this study. The rice plants and Ustilaginoidea virens strain were provided by the College of Biosystems Engineering and Food Science, Zhejiang University. In this study, the plants were inoculated with conidial suspensions according to previous research [Ustilaginoidea virens strain was cultivated with potato sucrose broth (PS) in an incubator shaker for five days at 150 rpm. at 28 °C. For spore inoculation experiments, about 1.5 mL of the conidial suspension (2 × 105 conidia mL−1) was injected into a single panicle using a syringe in the late afternoon at the booting stage, while the control plants were infected with double distilled water (ddH2O) according to previous study [Zheliang and 392 healthy, 430 slight, and 186 severe seeds of Xiushui were selected for analysis  was finally derived for further analysis. In order to collect multispectral images of rice seeds from visible to the near-infrared region for rice false smut (RFS) detection, a multispectral imaging system was developed in this study . Some keIn this study, the spectrum at different wavelengths was collected using a multispectral imaging system, and the individual reflectance can reflect the interaction between photons at specific wavelengths and plant materials. In order to ascertain the sensitivity of each wavelength to RFS, the principal component analysis (PCA) that used an orthogonal transformation was carried out by projecting the raw data into a new coordinate where the first component can describe the greatest variable of the original data on the first coordinate, the second greatest variance on the second coordinate, and so on . After tγ) and bandwidth (2δ) need to be optimized. The γ determined the tradeoff between minimizing the training error and model complexity, while the 2δ was the bandwidth of the kernel function. In this study, the radial basis function (RBF) was selected, and the grid searching technique was introduced for optimizing the bandwidth (2δ) and regularization parameter (γ). Linear discriminant analysis (LDA) described a categorical variable by linearly combining a set of features that could interpret two or more classes of objects [In order to develop an optical classification model for discriminating rice false smut (RFS) infected rice seeds from the healthy ones, three commonly used classification models were introduced to classify spectral features extracted from spectral images at six wavelengths. The best model was finally selected according to the best classification performance. Least squares-support vector machine (LS-SVM) has been proven capable of addressing not only linear, but also nonlinear multivariate analysis problems with a relatively fast way of mapping the original data space to a hyper-plane space using a kernel function ,21. LS-S objects , which w objects , chillin objects , and pat objects . In this objects . In this objects . The cla objects . Data an2R of 1.0000, 1.0000, 0.9996, 1.000, and 0.9999 for 460, 520, 660, 740, and 850 nm, respectively. Compared with linearities from these five wavelengths, it was relatively poor at 940 nm with a linearity of 0.9469. A good linearity of CCD can enable us to capture images using different exposure times of a CCD camera for the reference and samples with the aim of obtaining an optimal signal to noise (SNR).In order to understand the outperformance of CCD at six wavelengths, the linearities at 460, 520, 660, 740, 850, and 940 nm at a working distance of 18 cm were tested using a standard diffuse reference plate , respectively, as shown in Although original multispectral images can be corrected using a flat-field to eliminate the bias caused by the uneven distribution of light, it still needed to understand the illuminance distribution at every wavelength due to light from 12 LEDs emitting from different spatial positions  disease for the two cultivars were similar with a descending order of 740, 660, 520, 850, 460, and 940 nm for Zheliang and 740, 660, 850, 520, 460, and 940 nm for Xiushui, respectively. The value of principal component 2 was mainly determined by 740, 850, and 940 nm with positive values and 460, 520, and 660 with negative values. The direction of each wavelength in the vector graph indicted the effect on component value, while magnitude could serve as the impact of rice false smut (RFS) disease on reflectance. Zheliang, LDA and KNN achieved the same overall accuracy of 94.8%. Further analyzing the detection result of infected seeds from LDA and KNN, 85.4% of infected samples were correctly detected with the fact that 12 out of 82 infected samples were misclassified into the healthy class with a false negative rate of 14.6% for LDA, while 96.7% of infected samples were correctly detected with a false negative rate of 3.2% for KNN. Although the overall classification accuracy was the same between LDA and KNN, the KNN model achieved a relatively low false negative rate of the RFS detection, which is critical in finding as many infected samples as possible to prevent further infection. Compared with LDA and KNN, the classification accuracies for infected and healthy seeds of the LS-SVM model were in general better, as presented in Xiushui, the overall classification accuracies for LDA, KNN, and LS-SVM were in general poorer than those in Zheliang, which was reasonable because the disease in Xiushui was less severe, as shown in Xiushui in comparison with the LDA and KNN with the overall classification accuracy of 91.4% and false negative rate of 6.7%, respectively. These results imply that the LS-SVM was feasible for rice false smut (RFS) disease detection in rice seeds and the spectral resolution at these six narrow bands was enough to collect different information between healthy and RFS infected rice seed. It also needs to be mentioned that the classification accuracies for two different cultivars achieved in this study prove the feasibility of this multispectral imaging system for rice false smut (RFS) disease detection.The performance of the three classification models using six reflectances at 460, 520, 660, 740, 850, and 940 nm collected from a multispectral imaging system as model inputs was evaluated. Rice false smut (RFS) detecting results from LDA, KNN, and LS-SVM are summarized in Zheliang and Xiushui based on the LS-SVM model with the corresponding training dataset was proven, as shown in Zheliang than that in Xiushui. Therefore, we used data from Zheliang to train the LS-SVM model to identify RFS in Xiushui. In this study, the LS-SVM model from Zheliang was updated by adding a few samples from Xiushui. Xiushui, based on the model established from Zheliang, with the addition of different new samples from Xiushui selected by the Kennard–Stone (KS) algorithm. There was an increasing pattern of overall accuracies when the added samples increased from 1 to 3, and became relatively stable in the range of 4–8, indicating that the combination of two thirds of the whole Zheliang data with eight samples from Xiushui could provide enough variance about Xiushui in the new model. In this circumstance, the LS-SVM model with the values of γ and 2σ of 7748.7 and 0.04, respectively, was considered practically for RFS detection that could span both Zheliang and Xiushui. It obtained an overall accuracy of 90.3% with a false negative rate of 7.8% during the process of RFS detection in Xiushui, which was comparable to those from the model using its own data as the training set, as shown in Zheliang for RFS detection in different cultivars of rice seed, but also demonstrate that the availability of the developed multispectral imaging system only using six wavebands still achieved a good classification performance of RFS detection in two cultivars of rice seeds.In this study, the feasibility of detecting rice false smut (RFS) using multispectral imaging in two cultivars of Zheliang, respectively, and 91.4% and 6.7% for Xiushui, respectively, Additionally, the RFS discriminant model developed by Zheliang was successfully transferred to Xiushui with an overall accuracy of 90.3% and false negative rate of 7.8%. Overall, the multispectral imaging system developed in this study, combined with the LS-SVM model, can be implemented for fast, on-line RFS detection in rice seeds.In this study, we demonstrated the feasibility of a low-cost multispectral imaging system combined with chemometrics for rice false smut (RFS) detection in two different cultivars. Multispectral images at 460, 520, 660, 740, 850, and 940 nm yielded a significant difference between the healthy and RFS infected rice seeds. The LS-SVM model established from six reflectances could be used for discriminating RFS infected rice seeds from healthy ones with the overall classification and the false negative rate of 98.7% and 3.2% for


In the GH group, 25% (11/44) of the patients presented incidentally positive results. UPCR and total protein in 24 h urine specimens were increased in the GH group compared to the PE group. Receiver operating characteristic analysis showed a UPCR cutoff of 30 mg/mmol as significant for preeclampsia, while the sensitivity and specificity were 89%  and 100% , respectively. In the 24 h urine protein test, sensitivity and specificity were 80%  and 100% , respectively, for the cutoff value of 0.26 g/24 h. In comparison to the other commonly used tests here considered, UPCR determination is a reliable, relatively faster, and equally accurate method for the quantitation of proteinuria, correlates well with 24 h urine protein estimations, and could be used as an alternative to the 24 h proteinuria test for the diagnosis of preeclampsia.Preeclampsia affects 2–5% of pregnant women and is one of the leading causes of maternal and perinatal morbidity and mortality. We aimed to extensively evaluate proteinuria in women with preeclampsia and to determine the analytical sensitivity and specificity of and the cutoff values for urine protein-to-creatinine ratio (UPCR) and total protein in 24 h urine samples. This study included 88 women. We used the urine dipstick test, UPCR, and total protein measurement in a 24 h urine sample. The patients were divided in gestational hypertension (GH,  Preeclampsia (PE) is a multisystem disorder of pregnancy and is defined as the onset of hypertension accompanied by significant proteinuria after 20 weeks of gestation . PE typiAccording to the International Society for the Study of Hypertension in Pregnancy (ISSHP), preeclampsia, transient gestational hypertension, and gestational hypertension (GH) are characterized by the new onset of hypertension (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg) at or after 20 weeks of gestation ,6.Gestational hypertension is hypertension arising de novo after 20 weeks of gestation in the absence of proteinuria and without biochemical or hematological abnormalities. It is usually not accompanied by fetal growth restriction. Outcomes in pregnancies complicated by gestational hypertension are normally good, but about a quarter of women with gestational hypertension (particularly those who present it before 34 weeks of gestation) will progress to preeclampsia and have poorer outcomes [Preeclampsia is a multisystem disorder that manifests after the 20th week of gestation. It is characterized by hypertension and at least one of the following conditions: (1) proteinuria, (2) other maternal organ dysfunction, including acute kidney injury (AKI) , liver involvement  or alanine aminotransferase (ALT) > 40IU/L) with or without right upper quadrant or epigastric abdominal pain), neurological complications , hematological complications , (3) uteroplacental dysfunction , abnormal umbilical artery Doppler wave form, or stillbirth) [Proteinuria is not mandatory for a diagnosis of preeclampsia but is present in about 75% of cases. Rather, PE is diagnosed by the presence of de novo hypertension after 20 weeks of gestation accompanied by proteinuria and/or evidence of maternal acute kidney injury, liver dysfunction, neurological features, hemolysis or thrombocytopenia, and/or fetal growth restriction. Preeclampsia may develop or be recognized for the first time intrapartum or early post-partum in some cases .Moreover, PE is characterized by reduced renal perfusion and damage to glomerular basement membrane resulting in leakage of proteins in the urine. Irrespective of the cause of hypertension, the quantification of proteinuria in pregnancy is vital not only for making a diagnosis but also for predicting maternal and fetal outcomes. Normally, pregnant women excrete minimal amounts of proteins in the urine (up to 150 mg/day) because of renal changes that occur during pregnancy; however, proteinuria >300 mg/day is considered abnormal for pregnant women.Several methods for proteinuria evaluation are available in daily practice: (1) dipstick, (2) 24 h urine protein test, and (3) urine protein-to-creatinine ratio (UPCR). In the first method, urine test stripes are used. A standard test strip comprises up to 10 chemical pads that serve for the analysis of different parameters . The test can be read within 60–120 s after dipping the stripe in a urine sample. The chemical pads change color after being immersed in the sample, and the results are interpreted by comparison of the pad color with the colors presented in the dipstick analysis guide. A 24 h urine protein test requires a 24 h collection of a urine sample. Then, one aliquot is taken from the total volume of the 24 h urine sample and tested. The UPCR, like the dipstick test is based on the analysis of a random urine sample, but like the 24 h urine protein test, it provides quantitative results. The UPCR has been validated for use as an estimator of 24 h urine protein levels from one random urine sample by analysis of the protein-to-creatinine ratio.In clinical practice, proteinuria is assessed initially by automated dipstick urinalysis when possible; if this is not available, a careful visual dipstick urinalysis will suffice. If positive , the UPCR should be determined. A UPCR ≥ 30 mg/mmol (0.3 mg/mg) is abnormal. A negative dipstick test can usually be accepted, and further UPCR testing is not required at that time. Proteinuria is not required for a diagnosis of pre-eclampsia. Massive proteinuria >5 g/24 h) is associated with more severe neonatal outcomes [ h is assGenerally, the estimation of 24 h urine proteins has been considered the gold standard for quantitation of proteinuria . HoweverIn this study, we aimed to extensively evaluate proteinuria in women suspected of having GH or PE by comparing three available methods for protein testing: urine dipstick test, UPCR, and total protein in 24 h urine samples. We tested the analytical sensitivity and specificity and determined the cutoff values for UPCR in single mid-stream urine samples and total protein in 24 h urine samples. Moreover, we assessed biochemical parameters, such as serum creatinine, transaminases, and platelets, and evaluated their usefulness as additional markers of PE.This study included all patients with GH or PE (88 pregnant women) who were hospitalized between April 2015 and July 2017 at the Department of Obstetrics, Medical University of Gdansk, Poland. The patients were between 27 and 42 weeks of gestation with a singleton pregnancy with symptoms of GH or PE but without co-morbidities. Women with chronic secondary/essential hypertension, immunological diseases like Hashimoto’s disease, diabetes mellitus, pre-existing renal disease, intrauterine fetal death, multiple gestations, gestational diabetes and bacteriuria, multiple pregnancy, assisted reproductive technology in pregnancy, and premature rupture of membranes were excluded from the study. This study was approved by the Bioethics Committee at the Medical University of Gdansk (no. NKBBN/454/2014) and was conducted according to the principles of the Declaration of Helsinki. All participants provided a written informed consent to participate in the study.n = 44) and PE group (n = 44). The study flow diagram is shown in Based on clinical and laboratory evaluations, according to the ISSHP classification, the patients were divided into two groups: GH group (GH was defined as systolic blood pressure ≥140 mm Hg and diastolic blood pressure ≥90 mm Hg in a previously normotensive pregnant woman after 20 weeks of gestation without proteinuria or a sign of end-organ dysfunction. PE was diagnosed in patients with a high blood pressure (24 h respiratory rate records) and new-onset proteinuria, i.e., when resting blood pressure was ≥140/90 mmHg on two occasions that were at least 4 h apart, and significant proteinuria was detected in urine samples. In the absence of proteinuria, PE was diagnosed in women with hypertension in association with thrombocytopenia , impaired liver function , new development of renal insufficiency (elevated serum creatinine >1.02 mg/dL), pulmonary edema, new-onset cerebral or visual disturbances, or uteroplacental dysfunction, including FGR. FGR was diagnosed as fetal abdominal circumference/estimated fetal weight <10th percentile combined with pulsatility index in the umbilical artery >95th percentile, or pulsatility index in the uterine artery >95th percentile, or abdominal circumference/estimated fetal weight <3rd percentile, or absent end-diastolic flow in the umbilical artery [Proteinuria was assessed with the urine dipstick test, UPCR, and total protein in 24 h urine samples in each patient during hospitalization. The urine dipstick test was performed twice, and a positive result was considered significant. Subsequently, creatinine, AST, ALT, and complete blood count (CBC) were evaluated. Serum and urine biochemical parameters were assayed with Architect analytical system (Abbott) and CBC with Sysmex analytical system (Sysmex). The urine dipstick test was performed with the Iris urinalysis system (Beckman Coulter).p < 0.05. A receiver operating characteristic (ROC) analysis was performed to verify the diagnostic usefulness of the evaluated markers, and data are presented with 95% confidence interval (CI). Data are expressed as medians with interquartile range. Heat map and principal component analyses were performed in ClustVis, based on the Euclidean distance between clusters [All analyses were performed using Statistica 11.0 (Statsoft). Between-group differences were determined using nonparametric tests. Significance was set at clusters .p = 5.21 × 10−10; Mann–Whitney U test) and total protein in a 24 h urine specimen  were higher in the PE group. Each positive UPCR result corresponded to a significant total protein value in the 24 h urine specimen. The UPCR cutoff value of 30 mg/mmol was validated as significant for PE, based on the ROC analysis. While the area under curve was 0.96 , the sensitivity and specificity were 89%  and 100% , respectively. In the 24 h urine protein test, the area under the curve, sensitivity, and specificity were 0.94 , 80% , and 100% , respectively, for the cutoff value of 0.26 g/24 h  of the patients in the PE group, the urine dipstick test was falsely negative in the first test, while the second test revealed a positive result. In the GH group, 25% (11/44) of the patients had falsely positive results in the urine dipstick test; UPCR and 24 h urine test did not confirm the dipstick test results, but all three tests were performed. Moreover, UPCR  and hematocrit (HCT) ; however, the GH group had a higher hemoglobin (Hgb) concentration . Moreover, serum AST  and ALT  concentrations increased in the PE group compared to those in the GH group, but not serum creatinine .The CBC results showed that patients with PE and GH did not differ in whole-blood platelet (PLT) count , 57% , and 45% , respectively, for PLT; 0.630 , 61% , and 66% , respectively, for HCT; and 0.657 , 61% , and 59% , respectively, for Hgb. The area under curve, sensitivity, and specificity were 0.753 , 70% , and 66% , respectively, for ALT; 0.700 , 73% , and 61% , respectively, for AST; and 0.569 , 60% , and 48% , respectively, for creatinine . However, our data showed that 9% of patients were misdiagnosed as not having proteinuria using a single urine dipstick test, and a second sample testing was required. By contrast, the UPCR and total protein in a 24 h urine sample could fully distinguish patients with PE and GH, and a second test was not needed. Moreover, urine dipstick test results may vary depending on the maternal hydration status. Thus, even trace proteinuria may be reported as significant if the mother is dehydrated, and in contrast, proteinuria may be missed if the mother is overhydrated. In addition, the test readout could be altered depending on the test features (limit of detection and limit of quantification), alkalinity of the urine, and presence of infections. This is important, as proteinuria must be confirmed or excluded in pregnant women presenting with hypertension in order to diagnose PE.In our cohort, both UPCR and total protein in the 24 h urine test were validated as adequate for PE diagnosis. Both their sensitivity and their specificity were good enough to appropriately identify patients with PE or GH. However, while the 24 h urine test is considered the gold standard for proteinuria testing, the test itself is cumbersome, time-consuming, and prone to pre-analytical errors, which could in turn results in lack of compliance by and inconvenience to patients. Also, resting in the supine position during hospitalization may result in urine stagnation in the renal system, and the amount of collected urine may not reflect the actual 24 h volume. Hence, UPCR is a potentially useful marker of PE. As previously shown, UPCR was not affected by variations in urine concentration and the amount of urine excreted in 24 h . Our stuCreatinine was not adequate for PE diagnosis; its levels were comparable in patients with PE and with GH. Such a finding is apparently surprising, as one could suspect that protein loss due to altered renal filtration coexists with increased serum creatinine. Nevertheless, stable creatinine levels reflect adaptive renal capacities, and increased creatinine is more specific to kidney injury, while irreversible damage causes glomerulonephritis.Although AST, ALT, and PLT levels increased in the PE compared to the GH group, none of these values is useful for PE diagnosis based on the ROC analysis. Similar to creatinine, this finding may be because of early diagnoses in our cohort and lack of complications due to prolonged hypertension and proteinuria or signs of end-organ dysfunction. Collectively, the early diagnosis of protein loss due to hypertension prevented the subsequent development of full-blown preeclampsia with clinical symptoms and increased markers of tissue damage.The significant correlation between UPCR and proteinuria in 24 h urine samples is a noteworthy finding of our study. While the latter represents the gold standard for the diagnosis of proteinuria, it delays diagnosis by 24 h, is not well tolerated by the patients, and cannot be performed in an emergency room setting. When identifying markers of PE, not only sensitivity and specificity should be considered but also turnaround time from sampling to results, which may in turn lead to timelier decision-making, likely reduce patients’ anxiety, shorten the length of hospital stay, thereby minimizing the associated cost, and help “target” women with a real pathology for treatment.In practice, the 24 h urine protein measurement could mostly be replaced by a spot test determining urine protein/creatinine ratio, with a value ≥30 mg per mmol  representing significant proteinuria. This would eliminate the inherent difficulties in undertaking 24 h urine collections and speed up the process of decision-making ,18,19.The determination of the protein/creatinine ratio would be a valuable tool for the early diagnosis of preeclampsia if its accuracy is acceptable. It could prevent unnecessary hospitalizations and testing and allow earlier diagnoses . SeveralAlthough the 24 h urine collection is still accepted as the gold standard for the diagnosis of proteinuria during pregnancy, some authorities have suggested that the protein-to-creatinine ratio should be employed as the preferred method of quantifying proteinuria. It is recommended to use the 24 h urine test for subjects with an abnormal protein-to-creatinine ratio . In addiAlthough the 24 h urine test for total protein remains the gold standard for proteinuria evaluation, it has several limitations. The dipstick test has insufficient specificity and sensitivity; nevertheless, because of its simplicity and cost, it may still be used as a primary screening method but is not suitable for the quantitation of proteinuria. In comparison, the UPCR is a reliable, relatively faster, and equally accurate method for the detection and quantitation of proteinuria; it correlates well with the 24 h urine protein estimation and, thus, could be used as an alternative to 24 h urine testing for patients suspected of preeclampsia. Furthermore, the UPCR test can be performed in emergency room settings and is more clinically useful for the assessment of proteinuria in pregnant women with hypertension.


Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.Adhesive secretion has a fundamental role in barnacles’ survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle  Sessile marine organisms are bioinspiring for the development of new biomimetic materials and mechanisms. Barnacles’ attachment is very plastic, as they live holdfast to a variety of substrates, under a variety of hydrologic regimes, in oceanic or coastal habitats, submersed or intermittently immersed in intertidal zones, in protected overhangs, crevices, in the deep sea, or directly exposed to strong waves ,2. AdhesThe original model of adult barnacle’s underwater adhesion  describePollicipes pollicipes (Crustacea: Cirripedia) is found at the top of the peduncle core, formed of clusters of a single type of adhesive-secreting unicellular gland, mostly just below the mantle cavity, and a network of ducts that coalesce and carry the adhesive to the base of the peduncle  score calculated by MaxQuant a. The maP. pollicipes [The canonical barnacle’s cement proteins were not detected in the quantitative analyses of the cement gland at the proteomic level, except for CP100k a. On thellicipes  and autollicipes .In addition, other abundant proteins in the cement proteome were associated with “chemical cues” b. MULTIFProteins without annotation, uncharacterized, or just predicted were found to be abundant in the cement proteome . Some ofP. pollicipes cement proteome, to 38 previously identified, classified, and characterized cement proteins of various acorn barnacle species, gathered from NCBI and the literature, belonging to 8 different barnacle species . The analysis allowed observation of the clustering patterns of the unannotated proteins with the groups of proteins previously defined [Afterwards, unannotated and uncharacterized proteins were subjected to principal component analyses (PCA) to cluster with known cement adhesive proteins . PCA was defined   extracted by the PCA explained 46.4 % of the total data variation , allowing observation of the proteins’ grouping as a function of the relative amino acid composition . PC1 disd Kaplan , did notA. amphitrite protein, PCA situates it in G1  in the proximity of G1 , but in none of the cases in the proximity of the two other groups, G2 (surface coupling proteins of CP20k family) or G3 (bulk proteins of the families CP52k and −100k). Of the remaining 11 proteins, one of them, the most expressed of all (PP91716), was situated close to AE36k; 3 little expressed proteins  were located close to AE7k and AE22k; and the remaining 7 were found scattered among the three groups . One of the cement proteins picked from NCBI, AA52-3L, was misclassified according to the PCA analysis performed. Based on the relative amino acid composition of this it in G1 , but accit in G1 .P. pollicipes cement proteome that could not be annotated by homology nor a conserved domain found, and one new CP that was automatically annotated as being a CP52k-L. Among the latter, the 10 proteins that clustered with G1 surface-coupling proteins were found to be highly disordered (> 65% disorder), presented a great percentage of its structure in the form of loops (> 60%), and a very small percentage in the form of α-helices (< 10%); more than 60% of their residues were exposed, less than 5 % were intermediate residues, and less than 35% were buried; these characteristics together with the high protein disorder estimated, agreed with the G1 protein characteristics. Their isoelectric point, aromatic index, and the aliphatic percentage also fall in the range of those of G1 proteins. Regarding the other characteristics of cement proteins, these are presented in Pollicipes pollicipes was mainly composed of structural proteins and proteins involved in muscle contraction  family. Similar to our findings, three proteins of the HSP family were detected as significantly upregulated in the proteomic profile of juveniles from the barnacles phitrite . This faphitrite . Indeed,phitrite ,28. HSPsphitrite  and metaphitrite ,31,32, sOn the other hand, the absence of barnacle-specific cement proteins in the gland proteome, except for the CP100k, could be associated with the complexity related to handling this type of sample, affecting the detection of those proteins at the proteomic level. Factors related to concentration, precipitation, protein digestion, and dissolution compared to the major components of the samples (muscle proteins) could affect the absolute abundances. In this respect, the total number of shared proteins between the gland and cement proteome was relatively low. Note that around 50% of the total proteins identified in the cement proteome could be found in the gland proteome a, while Nonetheless, CP100k was represented in the gland proteome by a cluster of six proteins from the proteinGroup id 1217 . The leaP. pollicipes cement (96.2%), with CP52k being the most abundant of all at 60.3 %  and membranes, protease inhibitors, and proteases b. The bat 60.3 % b. The otns found . The prens found  were notous base ,37,38, ajaponica .P. pollicipes cement presented a highly varied composition of other proteins than the canonical barnacle-specific cement proteins, being the second most abundant protein family of the cement proteome (12 %), composed of molecules that function as chemical cues, the MULTIFUNCin, and the settlement-inducing protein complex (SIPCs). SIPC is a glycoproteic contact pheromone that triggers conspecific larvae to induce gregarious settlement, first isolated in A. amphitrite [P. pollicipes cement proteome. Its function there is not clear; it may function as an adhesive protein in adults as well, or it may have a pheromone function, like in barnacle cyprids, directing the conspecific larvae path along the peduncle to the substrate. In pedunculate barnacles, cyprid settle in adults’ capitulum shells or peduncle’s cuticle, walking down to the peduncle’s base in a couple of weeks [P. pollicipes cement proteins, indicating that it may play other roles in addition to being a chemical cue, possibly as adhesive, similarly to the cyprids. In this species, the SIPC’s adhesive role may be related to the binding to chitin, rather than to minerals of the shell and biomineralization as previously proposed for acorn barnacles [P. pollicipes’s chitinous cuticle tends to be thicker and prominent at the peduncular base, and SIPC may be one of the proteins that adhere to it. The origin of the SIPC found in the cement may be the gland, since it was also found there, although in a much smaller relative amount as compared to the cement.phitrite . It is ephitrite ,42. SIPCphitrite . In adulphitrite , simultaphitrite . SIPC waof weeks ,47. The of weeks ,14,48, barnacles , since iA. amphitrite SIPC shows up to 31% similarity to α2-macroglobulins (A2M), a broad-spectrum protease inhibitor also involved in the immune response in invertebrates [tebrates , thoughttebrates ,50. Varitebrates . If it itebrates .Balanus glandula MULTIFUNCin shares 78% nucleotide sequence homology with A. amphitrite’s SIPC, and it was proposed that the two proteins have a common evolutionary origin [P. pollicipes gland and cement proteomes, but in a much greater relative proportion in the cement, where it makes up nearly 9% of the whole cement proteome, three times more than SIPC. Its function there may have to do with settlement induction and, based on the combined evidence, possibly with adhesion and defense, similarly to SIPC, as they both evolved from A2M. The defense role of MULTIFUNCin in the cement may be against epibionts, as was proposed for B. glandula [MULTIFUNCin is a multifunctional glycoprotein cue that induces habitat selection by, and predation on, barnacles . Its expy origin . MULTIFUglandula . MULTIFUglandula , and theglandula , so it iP. pollicipes cement in a relative amount of 2%, in accordance with the previous findings in Chthamalus fragilis [P. pollicipes cement.Apart from A2M, which was only found at the cement gland in a minute relative abundance, a good amount (4.5%) of serine protease inhibitors (SERPINs) and other protease inhibitors, such as pacifastin, were found in the cement. Protease inhibitors’ presence in the cement makes a lot of sense, since the proteases of other species, namely micro- and macrofoulers, are a threat to cement integrity. Serine proteases and other proteases, including metalloproteinases and disintegrin, were found in fragilis . Evidencfragilis . This prfragilis ,56. The fragilis , was alsP. pollicipes cement at high relative concentrations. These enzymes are responsible for performing post-translational modifications of cement proteins. Peroxidases are involved in oxidizing phenolic cross-linking chemistry, and lysyl oxidase is involved in lysine/arginine protein cross-linking, reactions also observed in collagen’s and elastin fibrils’ cross-linking [A. amphitirite prefers phenolic chemistry over amino acids [Other oxidases, such as lysyl oxidase (5.5%) and other peroxidases, namely peroxinectin (1%), were present in the -linking . Its pre-linking . A peroxno acids . Similarno acids ,59,60.A total of 17 unannotated proteins accounted for around 24% of the cement proteome. Some of these proteins were also found to be highly expressed in the gland proteome, as well as in the gland transcriptome. Although CP19k was found among the annotated proteins, the presence of surface-coupling protein was relatively low, thus some unannotated proteins may be playing this function in the interface of bulk proteins and the substrate. As previously described, surface-coupling proteins are structurally disordered, which confers them elasticity ,62,63,64Among them, the PCA and the analysis of the physical and chemical parameters revealed that at least 9 of the 17 unannotated proteins showed most of the requirements to be considered new adhesive proteins. These new cement proteins are highlighted in gray in A. amphitrite [T. japonica TJ52k, classified in bulk proteins by PCA, do not match those of the cement protein group (G3). The degree of disorder is too high for a bulk protein, as well as the percentage of loops, and the reduced value of exposed residues. This may occur because the sequence of the protein analyses is incomplete [The secondary structure predicted for the protein AA_52k-L3 of phitrite  depositecomplete  and too Sacculina yatsui internal-specific secretory protein (issp-11), a member of hemolymph juvenile hormone-binding protein (JHBP) superfamily.Concerning other unannotated proteins not in the vicinity of G1, they are cement proteins that may be adhesive or not. If they are adhesive proteins, they belong to different adhesive protein families with a different amino acid composition and physico-chemical characteristics. Otherwise, they are new cement proteins but with an unknown function. Three of these proteins  grouped together between G2 and G3  and werePollicipes pollicipes (Crustacea: Cirripedia: Pedunculata) specimens  were collected on the rocky shore of Praia da Memória beach, Porto, Portugal, during low spring tide in August 2017. Individuals with undeveloped ovaries were selected owing to the proximity of the ovary to the cement gland to avoid contamination. Animals were brushed to clean epibionts, transported to the laboratory on ice, further swept with an ethanol-soaked cellulose cloth, and dissected upon arrival. The cement gland was located according to previous studies [Six immature  studies . The tis studies . Tissuesg, 30 min, RT). LC-MS/MS analyses of purified and desalted peptides were performed on a Dionex UltiMate 3000 n-RSLC system, connected to an Orbitrap FusionTM TribridTM mass spectrometer . Peptides of each sample were loaded onto a C18 precolumn , washed for 3 min at a flow rate of 6 µL/min, and separated on a C18 analytical column  at a flow rate of 200 nL/min via a linear 120 min gradient from 97% MS buffer A (0.1% FA) to 25% MS buffer B , followed by a 30 min gradient from 25% MS buffer B to 62% MS buffer B. The LC system was operated with the Chromeleon software  embedded in the Xcalibur software suite . The effluent was electro-sprayed by a stainless-steel emitter (Thermo Scientific). Using the Xcalibur software, the mass spectrometer was controlled and operated in the “top speed” mode, allowing the automatic selection of as many doubly and triply charged peptides in a 3-s time window as possible, and the subsequent fragmentation of these peptides. Peptide fragmentation was carried out using the higher energy collisional dissociation mode and peptides were measured in the ion trap (HCD/IT).Provided lysates were incubated for 30 min with 5 mM tris(2-carboxyethyl)phosphine (TCEP) at 56 °C. The solution was brought to 10 mM TCEP and 10 mM methyl methanethiosulfonate (MMTS) for 15 min to reduce and protect cysteine residues, respectively. Protein purification, protein digestion, and peptide purification were performed according to a slightly adapted single-pot solid-phase-enhanced sample preparation (SP3) protocol ,73. SequP. pollicipes custom proteins database using MaxQuant freeware (version 1.6.2.3) [P. pollicipes transcriptome. From these, 39,123  were annotated using a local BLAST with BLASTp program, against the non-redundant protein database , setting a cut-off e-value of 1e−3. The DATASETs S1, S2, and S3 were deposited at the Mendeley Data repository with the following Digital Object Identifiers: http://dx.doi.org/10.17632/bc65592ymt.1, http://dx.doi.org/10.17632/pgkf3mtb4m.1, and http://dx.doi.org/10.17632/92wsg4g3mt.1, respectively. It is noteworthy that this transcriptome was previously published [MS/MS raw data files comprising three biological replicates per sample studied, gland (DATASET S1) and cement (DATASET S2), were processed independently against a 1.6.2.3) . The proublished  and was http://bioinformatics.psb.ugent.be/webtools/Venn/).MaxQuant parameters for protein identification were: MS and MS/MS tolerances of 20 ppm and 0.5 Da, respectively; two missed tryptic cleavages were allowed; PSMs were accepted at a 1 % false discovery rate (FDR); and trypsin was selected for protein cleavage. The modification of cysteine by MMTS (methylthiolation) was set as a fixed modification, while oxidation of methionine and acetylation of protein N-terminus were chosen as variable modifications. Protein quantification was based on the approximate absolute protein abundance an intensity-based absolute quantification (iBAQ) score calculated by MaxQuant. Venn diagrams were used to identify the shared proteins among the majority proteins of each replicate and figures were built using an online free tool, available at the webserver of the Bioinformatics and Evolutionary Genomics Center  . Originahttp://web.expasy.org/protparam/), including molecular weight and isoelectric point, instability index, hydropathy, percentage of positive, negative and aromatic residues, and aliphatic index. Predictions on the secondary structure composition, solvent accessibility, and protein disorder were performed by PredictProtein [https://www.predictprotein.org/) from protein sequences by Meta-Disorder [P. pollicipes  deposited at NCBI and the literature. Only 20 amino acids were considered, for aspartic acid and asparagine were analyzed together, as well as glutamic acid and glutamine, since in some cases, CPs’ data delivered by the authors was in this form . Statistica  was used to perform the PCA analyses.Protein sequences found in the cement proteome without hit or annotations were blasted online against the non-redundant protein database (nr at NCBI), using automatic adjustment of the BLASTp program. Afterwards, proteins sequences were re-annotated according to the hit description or considering conserved domains. In case proteins remained without a hit or lacked a conserved domain, as previously described for most cement proteins, they were characterized using the ProtParam tool from EXPASY (tProtein  (https:/Disorder . It is aP. pollicipes. The gland proteome was found to be dominated by proteins involved in muscle, cytoskeleton, and some uncharacterized proteins, while minor components are involved in the stress response, detoxification, immunity, protein biosynthesis, protease inhibitors, and chemical cues. On the contrary, the cement is mainly composed of the barnacle’s adhesive proteins, unannotated proteins, enzymes, chemical cues, and protease inhibitors. The bulk proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Nonetheless, some of the most expressed proteins found lacked homology to any known protein. However, some of these unannotated proteins were found to be highly expressed both in the gland and the cement proteome, as well as at the transcriptomic level. Considering the lack of homology with known proteins, their amino acid composition, molecular weight, isoelectric point, and other chemical physical properties, we conclude that nine of them are surface-coupling adhesive proteins of G1, related to CP19k, as revealed by principal component analyses. Moreover, the low relative expression of known surface coupling indicates that those unannotated proteins could be playing a fundamental role in the bulk proteins–substrate interface. The other 12 unannotated proteins found in the cement may be adhesive or not, but their characteristics fall outside those of the adhesive protein groups characterized so far in the literature. More analyses must be performed in the future to validate the role of the unannotated proteins. The thorough quantitative description of the proteins analyzed, both in the gland and cement from the barnacle P. pollicipes, provides clues to understand the diversity of adhesive proteins and their function in cirripeds, serving as complementary information to update the barnacles’ cement adhesion model.This work revealed for the first time the whole proteome of the adhesion system of the barnacle


Chronic kidney disease (CKD) is one of the most common chronic non-communicable degenerative diseases and it represents an important risk factor for cardiovascular morbidity and mortality. The Mediterranean diet, in which extra virgin olive oil (EVOO) is the main source of vegetal fats, represents a nutritional-diet regimen that is useful for the treatment of CKD and its comorbidities. We tested two different EVOOs, characterized by a high (Synergy) and medium (Luxolio) content of minor polar compounds (MPCs), detected by HPLC-DAD-MS analysis, in 40 nephropathic patients, at a dose of 40 mL/day for 9 weeks. We evaluated the effects of these two EVOOs on renal function, body composition, oxidative stress, and inflammatory state, after 9 weeks of EVOOs consumption (T1) and after 2 months of wash-out (T2). We observed an improvement of renal function biomarkers , lipid profile, oxidative stress, inflammatory parameters  and in body composition at T1. These healthy effects were greater and persisted over time after the wash-out period in Synergy patients. The high MPC EVOO content seems to exert an antioxidant and anti-inflammatory effect in nephropathic patients and these protective actions are maintained over time. Chronic kidney disease (CKD) represents one of the leading chronic non-communicable degenerative diseases in the world. CKD is associated with high morbidity and mortality, in addition, the costs of renal replacement therapy (RRT) have increased significantly from 1960 up to today . FurtherAmong the typical products of an MD, a key food is represented by extra virgin olive oil (EVOO), which is its main source of vegetable fats. A total of 98–99% of the total weight of EVOO is made up by fatty acids, in particular, monounsaturated ones such as oleic acid, and a small percentage (1–2%) by minor polar compounds (MPCs). MPCs, especially of the phenolic variety, seem to be fundamental for the health effects exerted by EVOO. Among these, hydroxytyrosol, tyrosol, oleacin and oleocanthal are of particular importance ,15. In tEVOOs selected for this study were produced using biphasic pressing technology. The first oil selected was monocultivar Mignolo, produced in Tuscany  in 2019 . The second oil chosen was a mix of three cultivars of olives including Moraiolo (33%), Leccino (34%), and Mignolo (33%) produced in Tuscany  in 2019. The monocultivar Mignolo oil was obtained from a biosynergy and biodynamic crop that was made with leguminous species capable of fixing important nutrients in the soil, that have positively influenced the quality of the olive tree and therefore of the oil produced. The two selected EVOOs were extracted and then analyzed by HPLC-DAD-MS for the evaluation of MPCs content, according to the protocol already described in another study conducted by our research group .In the present clinical study, a group of 40 CKD patients   under coLUX and SYN were administered to nephropathic patients at a dose of 40 mL/day, for 9 weeks. In particular, the EVOOs were consumed raw, as a condiment for dishes. At baseline (T0), we collected the clinical history for each enrolled patient and we evaluated laboratory parameters, such as creatinine, azotemia, uric acid (UA), lipid profile, inflammatory and OS biomarkers, in addition to a body composition assessment. The same parameters were also examined at the two subsequent time-points: at time-point T1 (after 9 weeks of EVOO consumption) and at time-point T2 (after 2 months of wash-out period). The flow-chart of the study design is illustrated in The study protocol was declared as being compliant with the Helsinki declaration by the PTV Independent Ethics Committee. All enrolled subjects signed an informed consent at the point of enrollment.+, HCV+, inflammatory and/or infectious pathologies in the acute phase; BMI < 18 kg/m2; pregnancy and breastfeeding. During the period of study, the patients did not undergo therapeutic changes.The inclusion criteria were individuals aged between 18 and 80 years, both sexes after acceptance of informed consent. The exclusion criteria were presence of cancer in the active phase, HIV, HbsAgAll patients followed an Italian standard Mediterranean diet with a controlled protein intake according to CKD stage ,19. All Two questionnaires were administered to all nephropathic patients with the aim to exclude potential biases due to changes in eating habits or physical exercise during the study. PREDIMED (Prevención con Dieta Mediterránea) and IPAQ  questionnaires were performed at all time-points of the study . The first assesses the degree of adherence to the Mediterranean diet  and the In addition, to assess the polyphenols intake from other foods, we recorded a 24 h dietary recall at all time-points of the study, highlighting homogeneous polyphenols consumption between the patients, at each time point of the study .2).Anthropometric parameters of all CKD patients were registered according to standard methods . Body weFor the assessment of body composition, all nephropathic patients underwent bioelectrical impedance analysis (BIA). Resistance, reactance, impedance, and phase angle at 50 KHz frequency were measured using a BIA 101S instruments (Akern/RIL System-Florence). For evaluation of body composition, we considered the following parameters: total body water (TBW), intracellular water (ICW), extracellular water (ECW), body cell mass index (BCMI), fat free mass (FFM), fat mass (FM) and muscle mass (MM) . Not allMoreover, the waist and hip circumferences were measured and the waist to hip ratio was assessed, at all times of the study.At T0, T1 and T2, we performed the following laboratory and urinary exams: azotemia, creatinine, UA, electrolytes, sideremia, glycaemia, lipid profile, chemical–physical urinary examination, albuminuria on morning urine. Moreover, inflammatory parameters such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and interleukin (IL)-6 were monitored at all time-points of the study. All patients also underwent capillary sampling using CR4000 tool for the oxidative stress evaluation and for the assessment of antioxidant defense mechanisms. In particular, a free oxygen radical test (FORT) and a free oxygen radical defense (FORD) test were performed ,26. The Laboratory parameters were monitored by Dimension Vista 1500 , while the lipid profile was detected by standard enzymatic colorimetric techniques  and IL-6 was monitored by IMMULITE 2000 XPi Immunoassay System .All other parameters were analyzed according to standard procedures in the Clinical Chemical Laboratories of the University Hospital, PTV of Rome.All data were entered into an Excel spreadsheet  and the analysis was performed using the Windows Social Science Statistics Package, version 25.0 .The descriptive statistics consider the mean ± standard deviation for the parameters with normal distribution (after confirmation with histograms and the Kolgomorov–Smirnov test), while for the non-normal variables, they consider the median and the interval (minimum:maximum).t-test for normal variables and a Mann–Whitney test for the non-normal variables. Regarding occurrences (percentages), the chi-square test, possibly corrected by Fisher’s exact test, was performed. A p-value < 0.05 was considered statistically significant.The comparison for the normal variables was carried out with a one-way ANOVA, while the comparisons between treatment over time  were conducted with a paired The two selected EVOOs were analyzed by HPLC-DAD-MS for evaluation of the MPCs content, the results of the analyses are reported in Through the HPLC-DAD-MS analysis, it was possible to evaluate the content of single molecules. In particular, the values of hydroxytyrosol, tyrosol and derivatives were taken into consideration, as reported by the European Food Safety Authority (EFSA) EU No 432/2012 health claim. In fact, according to EFSA, it is necessary to consume 20 g/day of olive oil with a content of hydroxytyrosol and its derivatives of 5 mg, to have positive effects on health and to yield protection from oxidative stress .The oleocanthal and oleacin content was evaluated in both EVOOs, given the importance of their antioxidant and anti-inflammatory effects, as described in 2005 by Beauchamp . MignoloA total of 40 CKD patients under conservative therapy were recruited for the in vivo study. The patients were divided into two subgroups, homogeneous for age, gender, and BMI. The first group assumed 40 mL/day of EVOO SYN, the second group assumed 40 mL/day of EVOO LUX, for 9 weeks. The epidemiological findings of the two study subgroups and their homogeneity are reported in the During the study, the patients dropped out due to factors unrelated to side effects deriving from EVOO consumption, as reported in 2 vs. 37.7 ± 18.7 mL/min/1.73 m2; p = 0.0235) and this increase was maintained after the wash-out period , as reported in p = 0.0413), an albumin enhancement , an azotemia decrease , a triglycerides reduction  and a UA decrease  at T1. After 2 months of wash-out, the increase in albumin (p = 0.0041), the reduction in azotemia (p = 0.0389) and the reduction in UA (p = 0.0230) were maintained over time, as reported in p = 0.0084). Moreover, we also demonstrated a reduction in inflammatory status, as monitored by CRP , ERS  and IL-6  at T1. Only the reduction in CRP was maintained after 2 months of wash-out , while the ERS returned to basal levels at T2  and the IL-6 reduction was also maintained after the wash-out period  (The laboratory and urinary parameters monitored at T0 (baseline), after 9 weeks of EVOO consumption (T1) and after 2 months of wash-out (T2) in the two study groups (SYN and LUX), are reported in = 0.043) .p = 0.0442), a significant UA reduction  and an improvement in inflammatory status, highlighted by the reduction in CRP and ESR levels . After 2 months of wash-out, we observed an increase in CRP  and ESR  levels and a decrease in serum albumin  compared to T1.In the LUX group, we observed an albumin significant increase  after 9 weeks of SYN consumption, but this reduction was not maintained after 2 months of wash-out, as reported in The OS assessments of the two study subgroups are reported in p = 0.0391), an TBW increase , ICW enhancement , a reduction in ECW , an MM increase  and a BCMI enhancement , after 9 weeks of SYN consumption. After 2 months of wash-out, we highlighted that the significant variation in reactance and MM, in respect to the baseline, were maintained after wash-out period , as reported in 2 vs. 9.1 ± 2.9 kg/m kg/m2; p = 0.0296).The body composition assessments of the two study subgroups are summarized in p = 0.0033) after 9 weeks of EVOO consumption, which decreased after 2 months of wash-out .In the LUX group, we detected a significant increase in TBW . The improvement in albuminuria is important in the clinical management of CKD patients as it is a well-known biomarker of kidney damage and it is one of the main factors associated with the progression of nephropathy. CKD progression seems to also be related to the activation of renal and systemic inflammatory pathways, which induces alterations of the glomerular filtration barrier, with consequent podocyte damage and development of proteinuria ,54. The In our study, we observed a significant reduction in triglycerides in patients treated with SYN, as already highlighted by our preliminary data . This efInterestingly, in the two subgroups we also observed a decrease in UA values at T1, compared to T0. In detail, patients treated with SYN showed a statistically significant reduction in UA values, after 9 weeks of EVOO consumption, which lasted until the time-point T2. While, in the LUX subgroup, although a statistically significant reduction was observed at T1, this was limited to the period of EVOO intake. In fact, after the wash-out period, the UA concentration increased up to baseline values.CKD is associated with an increase in UA values due both to its reduced renal excretion and to its altered tubular transport in the nephron . The UA In our study, we confirmed the anti-inflammatory action of EVOO MCPs ,15,70. IThe observed increase in albumin values, noted in both treated subgroups at T1, is in agreement with previous studies ,75 and iFurthermore, only in patients who consumed SYN, we observed a significant reduction in OS, monitored by FORT. These data do not seem to last over time, as OS tended to rise again 2 months after the wash-out period, yet remaining lower than the baseline values. The antioxidant action is exerted by the EVOO polyphenols, as suggested by previous studies ,78. In pAs regards the body composition assessment—monitored by BIA—we, interestingly, observed a BCMI improvement in SYN patients at T1. In the literature, it has been highlighted that BCMI is a very sensitive index for monitoring MM changes, which, in our study population, may be related to uremic sarcopenia ,82,83. BIn addition, we detected an increase in reactance in the SYN subgroup; a parameter related to the cellular integrity and nutritional status of the patients. These data agree with the albumin increase highlighted in the study. In fact, both parameters express an improvement in the patient’s nutritional status .Finally, we demonstrated a TBW% increase in both subgroups (SYN and LUX) at T1 and, at the same time, an improvement in the ICW% to ECW% ratio only in the SYN subgroup. These parameters agree with the observed MM% increase, which is characterized by a high percentage of ICW .Our study shows that daily use of an EVOO with high MPC content seems to exert an important anti-inflammatory and antioxidant action in nephropathic patients. Therefore, once again, the pivotal role that nutritional-diet therapy plays in the clinical management of CKD patients and the consequential improvements in their quality of life, exerting positive effects on CKD signs and symptoms, has been highlighted. Thus, this study in nephropathic patients may lay the foundations for further randomized clinical trials that confirm the important role played by EVOO MPCs in the management of CKD.