Multiple biomarkers may predict short and long-term prognosis in patients with coronary heart disease, but their impact is limited when used in addition to established risk factors such blood pressure, cholesterol levels, diabetes mellitus, smoking as well as age and sex. Arteries are an integral part of the cardiovascular (CV) system. Arterial stiffness has been shown to be a predictor of cardiovascular events and mortality independent of traditional risk factors. It has also been shown that increased arterial stiffness may predict cardiovascular events in asymptomatic individuals without overt cardiovascular disease. Measuring arterial stiffness may, therefore, identify patients at risk at an early stage. Antihypertensive treatment has been shown to reduce arterial stiffness beyond its antihypertensive effect. Arterial stiffness could, therefore, be a surrogate marker of treatment that relates to prognosis. Arterial stiffness has mostly been used in research protocols, and its use as a prognostic indicator in clinical practice is still uncommon. Several methods exist that can determine parameters related to arterial stiffness, both local and in specific artery beds such as the aorta. In this brief review we present methods to evaluate arterial stiffness, their clinical utility, limitations and the advantages of a novel method, the Cardio-Ankle Vascular Index. Easier and more reproducible methods to evaluate arterial stiffness may increase the use of parameter as a risk factor for coronary heart disease in common clinical practice. Factors predicting outcome in coronary heart disease may have a causal relationship to the disease, such as hypertension and hypercholesterolemia. It may also be a marker caused by the disease condition itself, including compensatory mechanisms, expressing the severity of the disease, such as elevated natriuretic peptides, and left ventricular ejection fraction. It is important to identify those at risk in order to initiate preventive treatment and closer follow-up in those at higher risk. Multiple biomarkers have been evaluated as risk markers  but thesArterial stiffness is a strong predictor of cardiovascular events and all-cause mortality –7, also Arteries have an outer tunica adventitia consisting of fibro-collagenous tissue and the external elastic lamina. The innermost layer is the tunica intima with fibro-collagenous tissue, internal elastic lamina and endothelium. The large arteries, which consist of the aorta, brachiocephalic trunk, carotid and iliac arteries, have a thick middle layer, the tunica media, with smooth muscle fibers and numerous layers of concentric elastic fibers. The elastic nature of these vessels allows them to distend and act as a reservoir to accommodate blood ejected by the heart in systole. Arteries distal to these such as brachial, tibial, as well as the coronary arteries are the muscular arteries, where the tunica media has predominantly a thick layer of smooth muscle fiber . The vasDuring systole, the blood ejected by the left ventricle creates a wave propagating forward along the vascular wall of the large vessels distal to the heart. As the wave reaches the muscular arteries mismatched impedance results in a second smaller wave of reflected pressure traveling backwards. This wave reaches the aortic root in late systole or early diastole, augmenting aortic pressure aiding coronary flow –15. WhenWith aging, atherosclerotic changes occurs in the arteries resulting in increased stiffness. There is also an increase in wall thickness due to intimal thickening. Increased stiffness and an increase in wall thickness occur with age, also without atherosclerotic disease, due to depletion and fragmentation of elastin and the deposition of collagen in the media . PP chanWhen pressure increases within an artery it distends the artery. The relationship between pressure and diameter is not linear. Arterial stiffness (change in pressure/change in diameter (ΔP/ΔD) gradually increases with increasing pressure Figure . Whereasp = D(ΔP/ΔD), and compliance Cv. = 2(ΔD/ΔP)/D /D , 20. D ip and Cv have different values at different pressures P and are less useful as patient specific indices on arterial stiffness and elasticity.The parameters EStiffness parameter β, suggested by Hayashi et al.  providesThis can be written asβ is a measure of arterial stiffness not influenced by blood pressure at the time of measurement. However, β varies between vascular beds as the properties of blood vessels vary . The higThe gold standard for evaluating arterial stiffness has been Pulse Wave Velocity (PWV). This is determined by recording pulse pressure waves at two different points along the arterial tree, such as between carotid and femoral, and measuring the time taken for propagation along that distance.2 = (ΔP/ρ) (V/ΔV) in 1926. V denotes volume, ρ the density of blood, ΔP and ΔV are changes in pressure and volume, respectively.PWV is related to arterial stiffness. Several mathematical models and experimental studies have described this in detail , 24. BraThe volumes in the equation may be converted to area × length of a cylinder as follows:D = vessel diameter. ΔD = Increase in diameter. L = Length2-πL(D/2)2}/πL(D/2)2ΔV/V = {πL((D+ΔD)/2)2-(D/2)2}/(D/2)2= {(D+ΔD)/2)2+ 2DΔD + ΔD2 – D2)/D2= (D2)/D2 = 2DΔD/D2 + ΔD2/D2=  derived from 24-h ambulatory blood pressure measurements , 35. Theβ is a measure of arterial stiffness, not dependent on the blood pressure at the time of recording, but is the property of a local segment of the artery. We may say that PWV, which measures a longer segment of the arterial tree, represents the average of the local β values from the measured segment.2 (2ρ/Psys-Pdia) = Ddia/ΔD.Equation (2) can be written as PWVdia/ ΔD in equation (1) we get; β = PWV2 (2ρ/Psys-Pdia) ln (Psys/Pdia)Replacing DFrom this we can derive a new stiffness parameter independent of blood pressure at the time of measurement.Heart-femoral (hf) PWV incorporates information from the entire aorta, including the base of the ascending aorta. However, non-invasive pressure recording at the aortic origin is not possible. Hasegawa introduced a method to measure transit time from the aortic origin to the femoral artery  by simulba). T is the sum of these two  is practically identical to Here transition time (T) from the aortic origin to the ankle  is divided into two parts. The first is the time from the first heart sound to the start of the brachial PW  ln (Psys/Pdia)+ b.CAVI is defined as the stiffness parameter obtained by using ha-PWV determined as above. The actual equation is CAVI = a × (ha-PWV)Here a and b are two scale conversion constants to match the values of CAVI to Hasegawa's hf-PWV . The patA large study with 32626 Japanese subjects, with and without CV disease, aged 20–74 years, established a baseline score for CAVI . The stu1 receptor blockers, will effect CAVI measurements  is a method by which arterial stiffness can be evaluated and is independent of blood pressure at the time of measurement. It is easy to measure and the reproducibility is good. As expected, CAVI increases with age and reference values are higher in men. Studies have shown that CAVI is significantly related to coronary artery disease and parameters associated with atherosclerosis. CAVI could be used in clinical practice as a new independent risk factor for outcomes in coronary heart disease. CAVI could also be a good surrogate-endpoint to follow the effect of blood pressure treatment, and may reveal differences between drugs causing similar blood pressure reduction.Arterial stiffness is an independent prognostic indicator in coronary heart disease. CAVI, which integrates stiffness parameter β in the equation of aortic pulse wave velocity, renders a stiffness index independent of blood pressure at the time of measurement. It is easy to measure with good reproducibility. Studies indicate that CAVI is superior as a prognostic indicator compared to conventional methods of determining arterial stiffness such as bf- or ba-PWV. CAVI may, therefore, be an important and independent risk marker in patients with chronic and acute coronary heart disease.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Every effort was made to contact the authors in this regard, but without success. The Editor deeply regrets any inconvenience that this retraction has caused to the readership of the Journal or to the authors of the previously published paper.Following an investigation, the Journal was able to confirm that this accusation of plagiarism is well-founded. On those grounds, the Editor of


Background: Cardiac tamponade (CT) is a dreadful complication of laparoscopic antireflux surgery (LARS) with unknown incidence, and preventive measures are yet to be defined. Incidence during LARS with respect to usage/configuration of graft deployment is analyzed. Three-dimensional (3D) analysis of tack distribution provided anatomical insight to prevent cardiac injury.Materials and Methods: Data regarding the usage and configuration of graft deployment are retrieved from the prospective database. Grafting was “posterior” or “posterior + anterior.” Incidence of CT in all hiatoplasties is calculated. Tomography is reconstructed in 3D, showing the spatial distribution of the tacks. Tacks are numbered in the surgical video. Corresponding numbering is applied to the tacks in any particular tomography slice, utilizing the 3D images as an interface. A numbering-blinded radiologist is asked to identify the offending and the nonoffending tacks as the cause of tamponade. Tack-to-pericardium distances are recorded. Tacks having no measurable distance from the pericardium are regarded as offensive.Results: One CT occurred in 1302 consecutive LARS (0.076%). The incidence is 0% when “no” (379) or “posterior” (880) graft is used as opposed to 2.3% rate in “posterior + anterior” (43) grafting. The distribution of “offensive,” “nonoffensive but nearest,” and “safe” tacks followed a pattern. All offensive tacks belonged to the anterior graft fixation, which we referred as the critical zone.Conclusion: CT during LARS is rare, and associated with graft fixation anterior to the hiatal opening. Avoiding graft fixation to the critical zone may prevent cardiac injury. In addition, using the three-dimensional (3D) ability of tomography in conjunction with the surgical video, the exact distribution of all 16 titanium tacks with special reference to their measured proximity to critical structures  allowed detailed anatomical insight into a tack fixation-related heart injury. Findings of the incidence and anatomical study have practical surgical implications that may be useful to prevent iatrogenic CT.As a center specifically involved in LARS and using graft fixation selectively,12–14 Currently, we abandoned doing Nissen's in favor of Toupet's after our results demonstrated no antireflux benefit in contrast to a significant increase in complications with Nissen's.14 After the hiatal opening was partially closed by sutures, grafts were fixed in two distinct configurations by tacks, depending on the size of the defect. In all mesh augmentations, a U-shaped graft was cut and positioned through the back of the esophagus . Our standard approach in varying degrees of surgical severity has been reported previously.All grafts used are monofilament polypropylene  and all fixations are done by helical tackers , without exception.Data regarding graft usage and the configuration of the deployment were retrieved from our prospective database. Incidence of CT in all configurations was calculated.A 42-year-old woman was referred to our center with severe gastroesophageal reflux disease (GERD) 5 years after having an open Nissen's procedure without mesh augmentation elsewhere. She had a giant recurrent hernia and pH-meter proven reflux; manometry showed moderate dysmotility. Surgery took 3 hours because of adhesions, and a configuration 2 graft deployment was performed. Recovery was uneventful until the 6th postoperative hour when orthostatic hypotension (systolic 85–90 mmHg) with an increased heart rate (120–125/min), unresponsive to fluid challenge, became evident. There was no chest pain, her enzyme levels and electrocardiography were normal. As hypotension in resting supine position and shortness of breath became evident, a bedside echocardiography and tomography were performed. Both revealed CT, and tomography also identified several “tacks” as the cause . She waswww.liebertpub.com/lap). The video of the operation was reviewed, and starting from the first fixation, all tacks were numbered consecutively  repairs were done without graft augmentation, 880 (67.7%) had configuration 1 graft deployment, and 43 (3.3%) had configuration 2 graft deployment. The only tamponade occurred in the reported case, and there is strong evidence  that it The incidence of CT in entire series was 0.076%, and it was 0.108% in 923 mesh-augmented hiatoplasties. The incidence was 0% when no graft (379) or only posterior mesh augmentation (880) was used. In 43 patients with anterior graft fixation, the incidence was 2.3% (1/43).None of the other 10 tacks were found to be offensive as there was a clear measurable distance between the tack and the pericardium in all slices. The closest tacks to the heart were tack numbers 7, 12, and 1, and the shortest measured distances from them to the pericardium were 0.59, 0.79, and 0.86 cm, respectively. These were the nearest nonoffenders. The rest seven tacks, radiating inferiorly away from the heart, were anatomically irrelevant as far as the cause of tamponade was concerned.Among the six offenders, tack numbers 10, 11, 13, and 14 actually form a horizontal line, underlining the base of the heart from right to left . These fIn fact, the offender description relates to a particular area, corresponding to the zone covered by the anterior rectangular strip graft as demonstrated and referred as the “critical zone.”Among nonoffending tacks, aforementioned tack numbers 7, 12, and 1, which came less than 1 cm close to the pericardium, were all adjacent to the critical zone, again pointing out a particular area. Tack numbered 12 belonged to the anterior graft fixation. Although it was 0.79 cm close to the pericardium, tack number 12 was the only nonoffensive tack among seven tacks applied for the fixation of the anterior graft. Surgically, it was placed about 1 cm posteroinferior to the offending “anterior tack line.” Tack numbers 7 and 1 were the most proximally placed tacks at both sides of the arms of the U-shaped graft . They weThe shortest measurable distance between tack number 3 and the vena cava was 1.2 mm. The shortest distance between the tacks numbered 4, 5, and 6 and aorta was 0.63, 0.65, and 0.39 cm, respectively .8 Actual mortality rate can even be higher as some deaths go unreported in peer-reviewed journals, which was shown by Frantzides and Welle8 by cross-referencing the FDAs Manufacturer and User Facility Device Experience (MAUDE) data with the published reports. Differing from the vast majority of acute CTs wherein there is a chest trauma or another usual suspect, an unrecognized injury occurring during abdominal surgery under mechanical ventilation further complicates the management.CT resulting from perihiatal abdominal surgery represents a particularly challenging subset of traumatic CTs with extremely high mortality rates. Small case studies reported mortality rates between 37.5% and 66.6%.15 Nevertheless, 15 mmHg pneumoperitoneum and reverse Trendelenburg positioning of the patient affect anatomy. A stretched diaphragm will be thinner and should be closer to the pericardium. The helical tack has had a clear warning note attached since 2004, discouraging its use on surfaces with <4 mm in thickness.16 Recently, additional warnings contraindicated their use on diaphragm in vicinity to the pericardium.17The risk involved with tacking, stapling, or suturing the diaphragm is known because its thickness ranges between 1.5 and 5.4 mm and averages only 2.9–3 mm at the tendinous portion.18–26 Fixation of a graft to reinforce a hernia repair  was the primary reason of injury in 17 of the 23 reported cases. The literature also indicates helical tacks as the most common predators among all fixing devices as they were responsible from 13 of the reported tamponades,26 resulting in mortality in 4.23 Ironically, despite the mentioned warnings about the use of fixators near the diaphragm, neither the number of reported incidents nor the mortality rate seems to change18–26 (In contrast, a detailed PubMed search identified LARS and perihiatal diaphragmatic graft fixation as the leading causes of CT among all 23 reported cases occurring during upper abdominal surgery.nge18–26 .27 Although not a single mesh erosion became clinically evident or diagnosed during our entire series, we are unable to say that we have a 0% rate as an erosion may remain asymptomatic and, therefore, undiagnosed unless specifically searched for which was beyond the scope of this article.In addition to CT, the unsolved problem of mesh erosion, which debilitates a number of patients on the long run, is also a serious issue.28–31It is, therefore, reasonable to assume that the temptation to avoid “hernia” recurrence is the reason why surgeons continued to overlook the possible risks of fixing a graft to the diaphragm. This temptation is actually evidence based. Mesh augmentation is shown to decrease the recurrence rate when dealing with large hiatal hernias in all four randomized controlled trials.8 To our knowledge, only Frantzides mentioned the dangers of graft fixation anterior to the hiatal opening.Upon acting as the medical expert in two iatrogenic CTs due to a fixator injury, which were diagnosed at autopsy, Frantzides suggested that straight hernia staplers rather than helical tacks should be used as fixators, and they should be applied “primarily posteriorly.”The present radiosurgical study confirmed Frantzides's foresight by providing data on the anatomical vulnerability of the area covered by the anterior rectangular strip graft . In thisFrom a surgical point of view, the transition of tacks from being “offensive” to “nonoffensive and irrelevant” is quite abrupt and covers a horizontal area with a width of <1 cm, which can be referred as the “potentially critical zone” . This is11 stitches,19 and even a liver retractor,1 were reported to cause CT, all pointing out an anatomical constraint rather than the type of the offender. Skill of the surgeon may have a secondary role, but the thickness of the diaphragm will be the primary determinant of the outcome. Since our reported complication occurred during the surgeon's 1265th case, technical experience was surely not enough to avoid the injury once a tack was fired in the critical zone.The critical zone, therefore, represents an area, where there is no guarantee for the safe application of a tack, stapler, or a stitch, because as shown radiologically in the presented case, there might be no definable diaphragm in that area. Other fixators, including straight hernia staplers,Therefore, usage of any graft fixator within the critical zone must be abandoned. Furthermore, extreme caution is warranted even when there is an absolute need for stitching. Given the dynamics of real-time surgery, the nearest threat comes from the “potentially critical zone” and this zone should also be avoided. The “safe zone” definition is certainly arguable because a tack was only 1.2 mm from the vena cava, and others about 0.5 cm from the aorta. To our knowledge, no previous fixator-related great vessel injury has been reported so far, but obviously, great caution is warranted.1–11 is a common procedure worldwide as it is the only chance for cure in patients with erosive GERD. LARS, for its most part, is performed to allow symptomatic relief for a benign disorder, and, therefore, should never result in mortality as far as the risk/benefit ratio is concerned. In particular, any specific instrumental risk is unacceptable and must be prevented.LARS, the principal cause of CT resulting from abdominal surgery,8 excellently showed that even deaths related to graft fixation go unreported in the peer-reviewed medical journals. The unreported deaths will remain a problem, but also interestingly no previous report has provided information about the incidence of cardiac injury during LARS. This study revealed an overall incidence of 0.076% in 1302 consecutive LARS procedures. In mesh-augmented cases however, the incidence was 0.10%. It is noteworthy that no tamponade occurs when either no grafting or only posterior grafting was implemented. In 43 anteriorly grafted patients however, the incidence was 2.3%. The rest 42 patients who had an anteriorly tacked second graft were only lucky as they probably had a thicker diaphragm.Although it is known that the definition and reporting of a complication are easy at its extremes and become controversial in case of a minor problem, Frantzides and Welle26 The Society of American Gastrointestinal and Endoscopic Surgeons' report identified tacks as the second choice for graft fixation, following suturing, which were preferred by 23.9% and 56.4% of the participants, respectively.32 Strikingly, helical tacks are causing more deadly problems than more commonly used suturing. The first report that specifically warned about the danger of a helical tack during LARS was published in 2002.3 As stated, ironically, numerous reports have repeated the same warning until now.26Majority of the graft fixation-related CTs to date have resulted from a helical tack.Helical tacks are applied under manual pressure against the tissue, causing further thinning down, and their deployment happens in an “all or none” manner with very effective drilling capacity that should never be underestimated. In the critical zone, where a pounding heart applies counter pressure during a systole, “helical tacks” are extremely risky and their usage must be abandoned.In conclusion, CT during LARS is fortunately rare and there is reason to believe that it is avoidable provided that the specific anatomical constraint is totally addressed. Findings of the anatomical study suggest that avoidance of graft fixation anterior to the hiatal opening during LARS will certainly decrease, if not eliminate, the risk of highly mortal CT.


Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitroP. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression  in situ. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate  and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 μM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections.It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales  Pseudomonas aeruginosa lung infections in people with cystic fibrosis (CF) are a prime example. Within individual lobes of the CF lung, genetically antibiotic-susceptible and -resistant P. aeruginosa sibling bacteria coexist  to quantitatively measure gene expression in P. aeruginosa in an in vitro aggregate model system. Our findings suggest that HCR v3.0 could prove to be a useful tool for analyzing in situ bacterial gene expression in virtually any species in any context.Despite decades of research that has elucidated mechanisms of bacterial virulence, antibiotic tolerance, and antibiotic resistance, many infections remain impossible to eradicate. Phenotypic heterogeneity likely plays an important role in the failure of drugs and the immune system to clear chronic infections. Chronic  coexist . This liin vitro , 3. Likein vitro –7. Whilein situ hybridization method that includes a quantitative, enzyme-free signal amplification step to help visualize low-abundant RNAs  Fluorescence micrographs of P. aeruginosa FRD875 probed with Eub338 (green) and either Paerug1, Paerug2, or Paerug3 (magenta). (B) Fluorescence micrographs of S. aureus MN8 probed with Eub338 (green) and either Paerug1, Paerug2, or Paerug3 (magenta). Individual Paerug1, Paerug2, and Paerug3 probe pairs bound to P. aeruginosa but not S. aureus. Scale bars, 1 μm. Download FIG S1, TIF file, 2.6 MB.Individual Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02622-19.2FIG S2P. aeruginosa FRD1 was probed with just the odd initiator probe from the Eub338 HCR v3.0 probe pair with all of the odd initiator probes from the 20 pair algD probe set (top images) and with the odd initiator probe from the Eub338 HCR v3.0 probe pair with the 3 odd initiator probes from the Paerug1 to -3 probe set (bottom images). None of the odd initiator probes individually, or in groups, were capable of inducing fluorescence amplification with the fluorescent hairpin probes when used without the even probe pairs. Download FIG S2, TIF file, 1.7 MB.Initiators probes do not induce amplification when only one probe in the initiator pair is used. HCR v3.0 initiator probes come in pairs of odd and even initiators. Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02622-19.3FIG S3algD HCR analysis verifies that new P. aeruginosaalgD HCR probes are quantitative. Single-cell fluorescent signals for the PAO1 ΔalgD/pMQ72::algD complement strain with 0.10% l-arabinose inducer for algD probe set 1 (orange) and algD probe set 2 (magenta). Pearson correlations (r) and numbers of cells analyzed (n) for each micrograph are indicated. Pearson correlation coefficient for the 5th replicate increases from r = 0.84 to r = 0.95 if the single outlier in the lower right is excluded. Each scatter plot represents data from a unique micrograph after algD expression was induced with 0.10% l-arabinose. Download FIG S3, TIF file, 1.2 MB.Two-channel Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa specifically, using our previous HCR v2.0 probe sets as starting points . When only one probe from each probe pair was used, no fluorescence was observed, demonstrating the background suppression feature of HCR v3.0 probes  and 2 orders of magnitude lower for Escherichia coli and Staphylococcus aureus  and another HCR v3.0 probe set (3 probe pairs) to detect g points . P. aeruobes see . As expeobes see  while ths aureus , demonstP. aeruginosa cells within a population. According to Trivedi et al. . Because the HCR signal scales approximately linearly with the number of target mRNAs per cell    and S3, ographs) , 9. Noteographs) , and so P. aeruginosa alginate  and nitrate reductase (narG) gene expression in aggregates formed by mucoid (FRD1) and nonmucoid (PA14) strains. This approach was chosen for several reasons. First, measuring algD expression in situ is of interest because alginate is overproduced by mucoid strains in CF lung infections . As expected, the mucoid strain expressed algD more highly than the nonmucoid strain  , we grewd strain . Spatialnterface . In thisnterface . This isnterface  and Wessnterface  that foud strain  and was d strain . Analysihis size , 24. Wit centers  and G, aess narG  and G. T10.1128/mBio.02622-19.4FIG S4P < 0.0001, unpaired two-tailed t test). Download FIG S4, TIF file, 0.5 MB.ABBA nonmucoid and mucoid aggregates grow faster and are larger near the air-agar interface. Quantification of mean aggregate volumes (A) and rRNA HCR signals (B) per nonmucoid PA14 (open bars) and mucoid FRD1 (closed bars) ABBA biofilm aggregate at different depths below the agar surface in each ABBA sample in biological replicates 1 (left) and 2 (right). Each bar represents the mean aggregate volume or HCR signal per aggregate in a 50-μm bin depth below the agar surface, error bars indicate SEMs , algD (magenta), and narG (cyan) HCR v3.0 probes. Shown are biological replicates 1 (A) and 2 (B). Scale bars, 100 μm. Mean algD (magenta) and narG (cyan) HCR signal per aggregate at different depths below the air-agar interface for nonmucoid PA14 (C and E) and mucoid FRD1 (D and F) in biological replicates 1 (C and D) and 2 (E and F). Quantification of mean algD (H and J) and narG (G and I) HCR signals per nonmucoid PA14 (open bars) and mucoid FRD1 (closed bars) ABBA biofilm aggregates at different depths below the agar surface in each ABBA sample in biological replicates 1 (G and H) and 2 (I and J). Each bar represents the mean HCR signal per aggregate in a 50-μm bin depth below the agar surface, error bars indicate SEMs . Download FIG S5, TIF file, 1.6 MB.Alginate and nitrate reductase gene expression is generally higher in mucoid Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the algD relative to oxygen availability, we used a microelectrode to measure oxygen concentrations from 0 to 600 μm below the nitrate-supplemented LB agar surface in mucoid and nonmucoid ABBA experiments after 16 h of growth. Unexpectedly, the mucoid strain showed a modest increase in its oxygen consumption rate compared to that of the nonmucoid strain  of the agar, from 0 to 350 μm below the agar surface, and maximally at ∼75 μM oxygen . Values are normalized relative to PA14 algD expression after exposure to anoxic (0 μM) conditions. Mean values represent triplicate technical replicates from two independent biological replicates, error bars indicates SEMs. Download FIG S6, TIF file, 0.1 MB.Alginate gene expression is induced most highly in planktonic cultures by anoxia. Expression of Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa strains express alginate genes in hypoxic zones, which goes against the general thought that alginate gene expression is constitutive in mucoid P. aeruginosa. Furthermore, these experiments demonstrate the utility of HCR v3.0 for analog quantitation of bacterial gene expression in situ at spatial scales relevant to microbial assemblages. Going forward, it will be exciting to combine HCR with tissue clearing methods such as MiPACT  broth and agar. Bacterial cloning, ABBA experiments, HCR analyses, and oxygen measurements were performed as described previously , 25–28. 25–10.1128/mBio.02622-19.8TABLE S1Table S1, DOCX file, 0.1 MB.HCR probe target sequences. Download Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02622-19.9TABLE S2Table S2, DOCX file, 0.1 MB.Bacterial strains. Download Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02622-19.10TABLE S3Table S3, DOCX file, 0.1 MB.Primers. Download Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02622-19.7TEXT S1Text S1, DOCX file, 0.1 MB.Supplemental methods. Download Copyright © 2019 Jorth et al.2019Jorth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Ischaemia-reperfusion injury in kidney transplantation leads to delayed graft function (DGF), which is associated with reduced long term graft function. Remote ischaemic conditioning (RIC) improved early kidney graft function in a porcine model of donation after brain death and was associated with improved long-term cardiac outcome after myocardial ischaemia. This randomised, double-blinded trial evaluated the effect of RIC on kidney graft outcome in the first year, and examined the predictive value of a new measure of initial kidney graft function, i.e. the estimated time to a 50% reduction in plasma creatinine post-transplantation (tCr50).A total of 225 patients undergoing deceased donor kidney transplantation were randomised to RIC or a sham procedure performed prior to kidney reperfusion. Up to four repetitive cycles of five minutes of leg ischaemia and five minutes of reperfusion were given. GFR, plasma creatinine, cystatin C and neutrophil gelatinase associated lipocalin (NGAL) were measured at three and twelve months and estimated GFR was calculated using four different equations. Other secondary outcomes were identified from patient files.RIC did not affect GFR or other outcomes when compared to the sham procedure at three or twelve months. tCr50 correlated with one year graft function (p<0.0001 for both mGFR and eGFR estimates). In contrast, DGF i.e. “need of dialysis the first week” did not correlate significantly with one year GFR.RIC during deceased donor kidney transplantation did not improve one year outcome. However, tCr50 may be a relevant marker for studies aiming to improve graft onset.www.ClinicalTrials.gov Identifier: NCT01395719. Delayed graft function (DGF) is a consequence of ischaemia-reperfusion injury and a common complication after deceased donor kidney transplantation ,2. It isRemote ischaemic conditioning (RIC) protects against ischaemia-reperfusion injury in various tissues in both experimental and some clinical studies, particularly in the heart ,7, but aThe multi-centre, randomised, controlled clinical study CONTEXT  evaluatewww.ClinicalTrials.gov Identifier: NCT01395719) was previously described in detail ..13].2 95% CI 49–63, n = 40; vs. sham-RIC: 52 ml/min/1.73m2, 95% CI 46–59, n = 32; p = 0.43).This extended analysis showed that both the three month and one-year graft function were also similar, with no significant differences either in mGFR or eGFR between RIC and sham-RIC . Also, n2 95% CI 44–52, n = 106; vs. no DGF 52 ml/min/1.73m2 95% CI 45–58, n = 35; p = 0.39) at twelve months.The tCr50, which is a previously developed marker of early graft function for the CONTEXT study , correlaIn this randomised clinical CONTEXT trial, RIC did not improve GFR at one year after renal transplantation. Also, RIC did not affect the risks of graft loss, death, rejection or NODAT. This suggests that RIC does not offer any long-term protection in deceased donor kidney transplantation. At the same time tCr50, the primary endpoint of the CONTEXT study, was shown to correlate significantly with GFR at one year.To our knowledge, the CONTEXT study is the largest investigation to date for RIC in deceased donor kidney transplantation using a multicentre approach. The results were further strengthened by the randomised double blinded design as well as the use of both mGFR and P-cystatin C which are considered to provide more accurate estimates of GFR compared to eGFR based on P-creatinine alone.Transplantation from deceased donors is accompanied by more profound ischaemia reperfusion injury and an increased risk of DGF compared to living donor kidney transplantation. Still, our results are clear and in concordance with findings in living donor transplantation reported in the REPAIR study , which iThus, the positive short term effects of RIC reported by our group in a porcine donation after brain death transplant model  were notIt is possible that another explanation as to why our study failed to show a protective effect of RIC may be the timing of application of the RIC stimulus which could be important for the protective effects. Transplantation is a unique setting for the simple reason that the organ is not connected to the recipient until reperfusion. It is believed that RIC may provide a first window of protection conferred by humoral factors released from the conditioned tissue. It is possible that such factors were no longer in circulation at the time of reperfusion although this was only 38 (IQR 19–54) minutes after the end of the RIC procedures last cuff release . The sugIn addition our study demonstrated a weak, but highly statistically significant, correlation between tCr50 and both mGFR and eGFR at twelve months. No such correlation was demonstrated when comparing the incidence of DGF with GFR at twelve months. This may suggest that tCr50 is a more sensitive marker of the impact of early graft function on long term GFR than the commonly used definition of DGF, i.e. the need for dialysis within the first week post-transplantation. Thus, tCr50 although not simple to calculate  could beIn conclusion, our results indicate that RIC of the organ recipient does not offer any advantages in kidney transplantation either on short or one year outcomes. tCr50 is negatively associated with GFR at one year and may be used as a marker of early graft function with an impact on long term outcome.S1 Checklist(DOC)Click here for additional data file.


Green banana fruit is an important starch resource that consists of flesh and peel. The physicochemical properties of flesh starch have been widely studied; however, those of peel starch have hardly been studied, leading to the waste of peel. In this study, the physicochemical properties of the starches from the flesh and peel of green banana fruit were investigated and compared. The dry flesh and peel had 69.5% and 22.6% starch content, respectively. The starch had oval and irregular granules with eccentric hila. Their starches had similar bimodal size distribution; the volume-weighted mean diameter was approximate 17 μm, and the peel starch had a slightly smaller granule size than the flesh starch. The maximum absorption wavelength was higher in peel starch than in flesh starch. The apparent amylose content of flesh and peel starch was 21.3% and 25.7%, respectively. The flesh and peel starches both exhibited B-type crystalline structures and had similar relative crystallinity, short-range ordered degrees, and lamellar structures. The swelling power was similar between flesh and peel starches, but the water solubility was higher in peel starch than in flesh starch at 95 °C. The peel starch had a higher gelatinization temperature than flesh starch, but their gelatinization temperature range and enthalpy were similar. Both flesh and peel starches showed a diphasic hydrolysis dynamic, but peel starch had higher resistance to porcine pancreatic α-amylase hydrolysis than flesh starch. The contents of rapidly digestible starch, slowly digestible starch, and the resistant starch of flesh and peel were 1.7%, 4.3%, 94.1% and 1.4%, 3.4%, 95.2%, respectively, for native starch, and 73.0%, 5.1%, 21.9%, and 72.3%, 4.5%, 23.2%, respectively, for gelatinized starch. Starch is synthesized in amyloplasts or chloroplasts, consists of amylose and amylopectin, and exists as semi-crystalline granules in plants. Usually, starch is stored in cereal seeds, fruits, and some metamorphic roots and stems ,2,3,4,5.Musa of the family Musaceae. There are over 130 countries and regions producing bananas in the world, mainly in Central and South America and Asia [2 and produced 13.324 million tons of bananas [Banana is an evergreen herbaceous plant that belongs to the genus and Asia . In 2016 bananas . Green b bananas ,13. The  bananas ,15,16. A bananas ,18,19.Banana fruit consists of flesh and peel. To date, research on green banana fruit has mainly focused on the flesh, and contains the investigation of flesh flour and starch. The studies on flesh flour include the effects of different treatments including chemical, physical, and organic acid treatment on the composition, color, water-holding capacity, oil-holding capacity, thermodynamic properties, and static rheological properties of flour, the content and physicochemical properties of resistant starch in flour, and the production process of flour ,22,23,24In this study, starches were isolated from the flesh and peel of green banana fruit. Their morphology, structure, and some functional properties were investigated and compared. Our objectives were to disclose the physicochemical properties of starches from green banana peel and provide useful information for the utilization of green banana peel.The contents of soluble sugar and starch in the dry flesh and peel of green banana fruit were determined. The flesh had a slightly lower soluble sugar content (3.4%) than the peel (4.1%); however, the starch content was markedly higher in flesh (69.5%) than in peel (22.6%). The present result agreed with the previous research that the dry flesh of green banana contains approximate 75% starch . The staStarch granules isolated from the flesh and peel were observed with a polarized microscope under normal and polarized light . The fleThe granule size distribution of starch is shown in The iodine absorption spectrum of starch reflects the content and structural characteristic of starch components—amylose and amylopectin—and its maximum absorption wavelength (λmax) is determined by the chain length of amylose and amylopectin . The iodThe crystalline structure of starch can be detected by X-ray diffraction (XRD). Starches from different plant sources can be divided into A-, B-, and C-type sources according to their XRD patterns . The sta−1 and 1022/995 cm−1 are widely used to quantify the ordered degree and the proportion of amorphous to ordered carbohydrate structure in starch [−1 was similar between the flesh starch (0.75) and peel starch (0.78), and that of 1022/995 cm−1 had also no significant difference between the flesh starch (0.90) and peel starch (0.88), indicating that the flesh and peel starches had a similar short-range ordered structure in the external region of the granule. The ratio of 1045/1022 cm−1 of flesh starch in the present study was significantly higher than that of previous study, ranging from 0.55 to 0.60 in two banana varieties [Fourier transform infrared spectrometer (FTIR) is often applied for the determination of the short-range ordered structure of starch. The attenuated total reflectance (ATR)-FTIR spectrum can reflect the ordered structure in the external region of the granules . For then starch . The staarieties . In the arieties , the tra−1 is caused by the periodic arrangement of crystalline and amorphous lamellae, corresponding to the lamellar thickness [−1, respectively, corresponding to lamellar thicknesses of 8.79 nm and 8.71 nm. The peak intensity was similar between flesh (202.2) and peel starch (199.4). The present results indicated that the starches from flesh and peel had similar lamellar structures.Starch is made up of semi-crystalline granules and consists of alternating amorphous and semi-crystalline growth rings. The latter is formed by a lamellar structure that consists of alternating crystalline and amorphous regions. The lamellar structure can be measured by the spectrum of small-angle X-ray scattering (SAXS) . Figure hickness . The peahickness . The fle−1. Starch with high amylose content requires a higher temperature to destroy its internal structure, leading to the gelatinization temperature being positively related to amylose content [The thermal properties of starch were measured with a differential scanning calorimeter (DSC).  content ,40,42. TCurcuma longa, 13% for Canna edulis, 15% for Canna indica, 25% for Lilium lancifolium [Aspergillus niger amyloglucosidase (AAG). Generally, starch can be divided into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS), according to its digestion rate [The flesh and peel starches were in vitro digested by both PPA and ion rate . The digion rate  measuredion rate . The digion rate . The sliThe fruits of green banana were harvested from the farmland of Lingshui, Hainan Province, China in March 2017. The flesh and peel were separated manually and used as plant materials.g for 5 min, and the supernatant was transferred to a 100-mL volumetric flask. The precipitate was treated twice more with 80% ethanol, and the supernatant was transferred and made up to 100 mL for measuring the soluble sugar content. The residual precipitate was suspended with 3 mL of deionized water, and incubated in a water bath at 100 °C for 20 min. After cooling to room temperature, 3 mL of 9.2 M HClO4 was added to the sample for 15 min. The sample was centrifuged at 5000× g for 5 min, and the supernatant was transferred to a 100-mL volumetric flask. The precipitate was treated twice more with 6 mL of 4.6 M HClO4, and the supernatant was transferred and made up to 100 mL for measuring the starch content. The soluble carbohydrates in the above supernatants were determined using an anthrone–H2SO4 method, and converted to the contents of soluble sugar and starch in flour.The fresh flesh and peel were immediately dried at 110 °C for 3 h and 80 °C for 2 d. The dried flesh and peel were extensively ground into flour in a mortar and pestle, and then passed through a 100-mesh sieve. The contents of soluble sugar and starch in flour were determined according to the experimental method described by Gao et al. [w/w) using a home blender . The resultant slurry was squeezed through four layers of cheesecloth and sieved through a 100-mesh and 200-mesh screen. The starch suspension was left overnight at 4 °C. The precipitated starch was washed three times with NaOH aqueous solution , three times with deionized water, and twice with anhydrous ethanol by centrifugation. During water washing, the dirty gel-like layer on top of the white starch precipitate was carefully scraped off and discarded. Finally, the starch was dried at 40 °C, ground into powder, and passed through a 100-mesh sieve.The starches were isolated from fresh flesh and peel according to the methods described by Waliszewski et al.  and Fan The 10-mg starch was suspended in 1 mL of 50% glycerol, and a 10-µL suspension was placed on the microscope slide and covered with a coverslip. The morphology of starch granules was observed and photographed using a polarized microscope  under normal and polarized light. The starch granule size was analyzed using a laser diffraction particle analyzer  according to the method described by Cai et al. [The 10 mg of starch and 5 mL of dimethyl sulfoxide containing 10% 6 M urea were added to a 10-mL glass tube, and then incubated in a water bath at 95 °C for 1 h with interval vortexing. After cooling to room temperature, 1 mL of sample solution and 1 mL of 0.2% iodine solution were added and made up to 50 mL in a volumetric flask with deionized water. The sample was held in dark for 20 min. The iodine absorbance spectrum was scanned using a spectrophotometer . The AAC was determined from the absorbance at 620 nm.The crystalline structure of starch was investigated using an X-ray powder diffractometer , following our previously described method . Briefly−1 to 800 cm−1 with 4 cm−1 resolution and 64 scans. The spectrum was corrected by a baseline between 1200 cm−1 and 800 cm−1; then, the deconvolution was performed using Resolutions Pro with a Lorentzian line shape, a half-width of 19 cm−1, and a resolution enhancement factor of 1.9.The short-range ordered structure of starch was measured using an FTIR spectrometer  with a deuterated triglycine sulphate (DTGS) detector equipped with an ATR cell following our previously described method . Brieflyg for 5 min, and the starch precipitate was sealed in a sample cell to prevent dehydration. The sample was measured using a SAXS instrument  equipped with a Vantec 2000 detector and pinhole collimation for point focus geometry. The X-ray source was a copper rotating anode (0.1 mm filament) at 50 kV and 30 W, and the Cu Kα radiation wavelength was 1.5418 Å. During analysis, the sample was under vacuum to avoid the air scattering. The SAXS dataset was analyzed using DIFFRACplus NanoFit software . The spectrum parameters were measured using the simple graphical method [The lamellar structure of starch was measured following our previously described method . Brieflyl method .g for 10 min. The weight of the original starch was recorded as W1, the starch weight in supernatant was determined by the anthrone–H2SO4 method and recorded as W2, and the mass of precipitate obtained after centrifugation was recorded as W3. The swelling power was obtained by W3/(W1 − W2) × 100%, and the water solubility was obtained by W2/W1 × 100%.The swelling power and water solubility of starch were measured according to the method described by Lin et al. , with soThe 5 mg of starch was accurately weighed in an aluminum pan , and then added to 15 µL of deionized water. The sample was mixed, sealed, and held at 4 °C overnight. After balancing at room temperature for 1 h, the sample was heated from 25 °C to 130 °C at 10 °C/min using a differential scanning calorimeter 3, 5 mM of CaCl2, 50 U PPA). The sample was incubated in a ThermoMixer C  at 37 °C and 1000 rpm for 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, or 48 h. After reaction, the sample was centrifuged at 8000× g for 5 min, and the soluble sugar in the supernatant was measured using an anthrone–H2SO4 method and converted to the hydrolyzed starch.The starch was hydrolyzed by PPA  according to the method described by Gao et al. , with so3, 5 mM of CaCl2, 4 U PPA , 4 U AAG ). The sample was incubated in a ThermoMixer C  at 37 °C and 1000 rpm for 20 min or 2 h. After reaction, the sample was immediately transferred to a 10-mL glass tube with 2 mL of 50% ethanol and 240 µL of 0.1 M HCl, and centrifuged at 8000× g for 5 min. The glucose content in the supernatant was measured with a glucose assay kit  and converted the degraded starch. The RDS, SDS, and RS were obtained according to the degraded starch within 20 min, between 20 min and 2 h, and after 2 h, respectively.The digestion properties of native and gelatinized starches were determined according to the previous method described by Lin et al. , with sot-test.All of the data in tables are given as means ± SD and analyzed with Student’s The dry flesh and peel of green banana contained 69.5% and 22.6% starch content, respectively, indicating that they are an important starch resource. Flesh and peel starches had oval and irregular granules with eccentric hila. The granule size of peel starch was slightly lower than that of flesh starch. The λmax and AAC were higher in peel starch than in flesh starch. Both flesh and peel starches had B-type crystalline structures and similar relative crystallinity, short-range ordered structures in the external region of the granules, and lamellar structures. The swelling power was similar between the flesh and peel starches, but the water solubility of peel starch was higher at 95 °C than that of flesh starch. The gelatinization temperature was higher in peel starch than in flesh starch, but their gelatinization enthalpy was similar. The digestion properties of native and gelatinized starches were similar between flesh and peel starches, except that the peel native starch had slightly lower SDS and higher RS contents than flesh native starch. This study could provide important information for the applications of the peel starch of green banana fruit.


Bread is categorized as having a high amount of rapidly digested starch that may result in a rapid increase in postprandial blood glucose and, therefore, poor health outcomes. This is mostly the result of the complete gelatinization that starch undergoes during baking. The inclusion of resistant starch (RS) ingredients in bread formulas is gaining prominence, especially with the current positive health outcomes attributed to RS and the apparition of novel RS ingredients in the market. However, many RS ingredients contain RS structures that do not resist baking and, therefore, are not suitable to result in a meaningful RS increase in the final product. In this review, the structural factors for the resistance to digestion and hydrothermal processing of RS ingredients are reviewed, and the definition of each RS subtype is expanded to account for novel non-digestible structures recently reported. Moreover, the current in vitro digestion methods used to measure RS content are critically discussed with a view of highlighting the importance of having a harmonized method to determine the optimum RS type and inclusion levels for bread-making. Carbohydrates are the most important source of dietary energy for humans  , with stBesides lacking the nutritional components from the whole grain fraction, white bread is categorized as having a high amount of rapidly digestible starch. This is the result of starch gelatinization produced as a consequence of the high temperatures that the dough reaches during baking ≥70 °C) at relatively high-water content (≥35%) ,7. In fa0 °C at rIn view of the large consumption of daily white bread and the health benefits associated with higher dietary fiber consumption , the enrResistant starch (RS) is defined as the starch portion that escapes digestion by human enzymes in the upper part of the gastrointestinal tract, entering the large intestine where it can be partially or fully fermented by colonic microflora. The main health outcomes of RS consumption can be categorized mainly based on a modulation on the glycemic response, body weight control, and bowel health. However, this review is not intended, by any means, to provide deep insights into the complex effects of RS consumption on specific metabolic responses and health benefits, which has been previously revised elsewhere ,25,26,27According to its definition, RS should be predicted by physiological (in vivo) techniques , such asMany in vitro assays for RS determination are variations on Berry’s  modificaIn 2002, McCleary and Monaghan  also devRS can also be measured following the procedures used for dietary fiber determination. However, attention should be paid on the methodology used because some RS sources can be underestimated. Thus, the Prosky  and Lee RS is usually categorized following the RS classification given by Englyst, Kingman, & Cummings ; EerlingThe resistant digestion property of RS can be the result of its confinement within the intact plant cell  and/or the food matrix. Overall, the role of cell walls in limiting starch digestion is based on three mechanisms ,55,56,57The presence of whole or partly milled grains and seeds has been reported to decrease the glycemic index of breads ,61. HoweBesides plant cell walls, storage proteins from certain plants, such as those from wheat (glutenin and gliadin), maize (zein), and sorghum (kafirin), have the ability to form disulphide bonds that result in a continuous layer around starch granules upon cooking, and in a slowdown of starch digestion ,66. In aStarch usually gelatinizes in the range of 54 to 76 °C at ≥20% water . TherefoRS2 has been found in ungelatinized tubers, particularly in potato, as well as in starchy fruits, such as green banana, both in vitro  and in vIn general, the addition of RS2 ingredients may result only in a moderate increase of RS in the final bread, as gelatinization will destroy their semi-crystalline granular structure. This moderate increase will be the result of remaining ungelatinized granules in the crust, which represents a significant, but lower portion of the bread slice. As an example, Roman, Gomez, et al.  observedAfter gelatinization, which results from baking, dispersed starch molecules begin to re-associate upon cooling, forming tightly packed structures stabilized by hydrogen bonding that are more resistant to digestion . The resIn contrast to amylose, the branched structure of amylopectin is less prone to retrograde, needing a longer time for the formation of double helical structures . RetrogrStarch resistance can also be created by the inclusion of chemical structures along starch chains. The resistance to digestion of chemically modified resistant starch (RS4) is dependent on the type and extent of the chemical modification, mostly consisting of dextrinization, etherification, esterification, oxidation, and/or cross-linking .The mechanisms responsible for the enzymatic resistance of RS4 have been revised elsewhere . It is oRS4 from different starch sources are a widely commercialized RS ingredient, although little information exists from the manufacturer about the nature and level of these modifications. Multinational companies providing RS4 from potato, tapioca, wheat, and/or high-amylose maize include Ingredion , RoquettAmylose can form inclusion complexes with lipids, and these complexes have been shown to be more resistant to digestion . AmyloseTwo forms of complexes can be distinguished depending on their thermostability: Type I amylose–lipid complexes that melt at about 95–105 °C (less ordered structures), and Type II  melting at about 110–120 °C ,118. Although there are no commercially available sources of RS5 in the market, amylose–lipid complexes can also reform upon baking, provided there are lipids in the formula. In this regard, most gluten-free breads, which are mostly made with maize starch and rice flour, incorporate some source of lipid/fat in the formula, to enhance the crumb softness and juiciness, as it tends to be excessively dry . MeanwhiIt is worth noting the ~50% reduction of postprandial blood glucose and insulin levels of breads containing 60% (flour basis) of a developed RS5 containing ingredient compared with the control white bread . These aAlthough bread-making varies widely around the world, the four basic ingredients are flour/starch , water, yeast, and salt. Processing conditions include kneading, proofing, and baking. Inclusion of RS ingredients in the formula is usually given by replacement levels of the starchy material by the RS ingredient. Most investigations approached the RS enrichment of breads using commercial RS2 and RS3 ingredients, but only some studies included the RS content in the final product. This is critical as baking will critically alter the type and amount of RS. For this reason, in High amylose starches, usually from maize, in both granular (RS2) and retrograded (RS3) form, are among the most used commercial RS ingredients in bread-making. They are widely available from many companies including Ingredion, Roquette, Cerestar, and SunOpta Ingredients. Tapioca rich in retrograded amylose (RS3) has also been investigated, which can be purchased from Cargill. RS2 from green banana starch or flour has also been evaluated in bread. The demand for banana starch/flour (RS2) is on the rise, and companies like Chiquita (Costa Rica), Livekuna (Canada), International Agriculture Group (United States), and Natural Evolution  commercialize a wide range of banana starch/flour ingredients with elevated RS content (~40–50%). On the one hand, it must be brought into attention that, converse to RS2 from high-amylose maize and RS3, RS2 from green bananas is not heat-stable and will not resist baking, that is, there will be a significant fraction of RS that will be lost during baking ,122. It The targeted amount of RS ingredient during formulation depends on the starch being used and the desired RS level in the bread. Normally, percentages of replacement have been reported within 5–30%, which resulted in breads with final RS content being dependent on the method of analysis. The RS content in both ingredients and breads was, in some cases, quantified by the AOAC Method 2002.02 and modifications of the Englyst procedure. However, AOAC official methods 985.29 and 991.43 were also used, which can lead to underestimations of the RS content . TherefoThe incorporation of RS ingredients into bread formula also brings about differences in the physicochemical and organoleptic properties of the bread . The flaIn addition, other than the nature of RS ingredients, processing conditions may also influence the formation of RS in bread. Baking under low-temperature and a long-time period significantly resulted in higher amounts of RS in bread than in those baked under higher-temperature and shorter time ,135,136.In some studies, the amount of water added to each formulation was adjusted based on the water binding capacity of starches as determined by farinographic analysis or elastic modulus ,138,139.RS generally meets the criteria to be defined as “dietary fiber” by the comprehensive dietary fiber definition adopted by the CODEX Alimentarius Commission . HoweverCurrent regulations also identify the potential physiological benefits of RS. The European Food and Safety Authority (EFSA) approved the health claim, “Replacing digestible starch with resistant starch induces a lower blood glucose rise after a meal”. However, this claim can be only used when the final RS content in the food is at least 14% of the total starch . On the The development of breads rich in RS and acceptable quality attributes could have a positive impact on the modulation of the glycemic response, the control of body weight, and the improvement of bowel health of bread consumers. The growing evidence of the positive health outcomes attributed to RS is leading to the apparition of novel RS ingredients in the market for bread-making, whose incorporation may seem the most logical and easy strategy to increase the RS content in breads. However, it must be noted that not all RS ingredients preserve the RS property during baking. It is thus paramount to understand the structural basis for their resistance to digestion and hydrothermal processing, which is often disregarded. This review concludes that high amylose starches, both native (RS2) and processed (RS3), are the most suitable RS ingredients for bread making in terms of RS preservation during baking and a lower detrimental impact on bread texture. However, their level of inclusion must be carefully selected. Another issue that this review addresses is the lack of harmony in RS values, which is the result of using different in vitro methods, some of which do not account for all types of RS structures. This outlook is changing though, as AOAC Method 2002.02 or any of its extensions, such as AOAC Methods 2009.01/2011.25, are adopted by many researchers from different nationalities.


Hybrid visceral-renal debranching procedures with endovascular repair have been proposed as a less invasive alternative to conventional thoracoabdominal aortic aneurysm or dissection (TAAA or TAAD) surgery. Up to now, there has been no information about the hemodynamic effects of retrograde visceral reconstruction (RVR) for Crawford type II/III/IV TAAA patients undergoing hybrid treatment. The aim is to provide insights in the abnormal hemodynamics of RVR from unilateral or bilateral common iliac arteries (CIAs). Idealized three-dimensional AAs with RVR from unilateral CIA or bilateral CIAs were generated and computationally simulated. The results show that RVRs from CIA lead to a dramatic decrease in flow to the visceral organs compared with a healthy AA and that the anastomosis region is most dangerous to graft occlusion and the initiation of an aneurysm. In addition, compared with a quar-furcated graft, the employment of bilateral bi-furcated grafts have better performance in terms of the wall shear stress (WSS) and flow filed but result in less flow to the celiac and mesenteric arteries. This study has revealed the potential risks after an RVR operation, and points out the advantages and disadvantages of different approaches for the visceral reconstruction, which the vascular surgeons are not fully aware of. According to our results, bilateral bi-furcated grafts are recommended to the TAAA patients when the CIAs are unique inflow sites for visceral reconstruction. A precise plan with patient specific for TAAA or TAAD will be designed for better long-term outcome. There are three operation treatments for TAAA or TAAD: open repair, endovascular repair and hybrid surgery. The hybrid procedure combining open surgical reconstruction of the visceral artery and endovascular aneurysm exclusion was first reported by Quinones et al. Being aThe majority of patients of hybrid repair underwent RVR from the aortic bifurcation or CIA,3 to visMoreover, when the normal CIAs are the only inflow sites for RVR, abdominal visceral arteries may be constructed by one quar-furcated graft from unilateral CIA or bi-furcated grafts separately from bilateral CIAs. VasculaIn recent years, numerical investigations have been used increasingly by researchers seeking to understand vascular hemodynamics. Many researchers have shown that computational fluid dynamics (CFD) can faithfully capture the physics of various vascular diseases including abdominal aortic aneurysm, cerebral aneurysm, aortic dissection, artery stenosis etc–15. In aAccordingly, this manuscript focuses on the hemodynamic influence of RVR on the aorta with Crawford II/III/IV TAAA using the CFD method described in our last paper. This woThe simple procedures of RVR with unilateral and bilateral CIA are:For unilateral CIA, one quar-furcated graft was constructed, and the main body side of a quar-furcated graft was anastomosed end-to-side to a unilateral CIA with a 5–0 Prolene suture to serve as the visceral inflow blood source. The four limbs of the graft were respectively anastomosed, end-to-side, to the right renal artery, superior mesenteric artery (SMA), celiac artery (CA) and left renal artery (LRA) with a 6–0 Prolene suture.For bilateral CIAs, two bifurcated grafts were chosen, and the main body side of one bifurcated graft was anastomosed end-to-side to a right CIA that served as the visceral inflow blood source. The two limbs of the graft were respectively anastomosed end-to-side to right renal artery (RRA) and CA. The other graft was constructed from a left CIA to SMA and LRA .The bifurcated vessel prostheses (Gore-TEX) of 16mm×8mm were actually selected for the bypass graft of RVR based on the experience in the West China Hospital. The vessel prostheses (Gore-TEX) of 16mm×8mm were more suitable to Asian populations than the prostheses of 18mm×9mm because of the smaller diameter of arteries in Asian populations. FurtherThe model geometry was built using software Pro/E 5.0 . Based on the postoperative computer tomography angiography (CTA) or DSA of patient with TAAA, the norIn the current study, the blood was assumed to be an incompressible, laminar, steady, homogenous and Newtonian fluid, and the corresponding governing equations are given asp represent, respectively, the fluid velocity vector and the pressure. ρ and μ are the density of 1050kg/m3 and dynamic viscosity of 3.5×10−3kg/m⋅s.Where m/s. Due to the clinical fact that the perfusion pressure of the visceral arteries are consistent if no artery stenosis happens, namely, the perfusion pressure for the visceral organs remain unchanged before and after the operation. So we first calculated the corresponding pressure drop from the supraceliac artery to each visceral organ in terms of the flowrate ratio for a healthy AA model, then we applied the same perfusion pressure to the corresponding visceral organ for the RVRs. In another words, assuming that the pressure at the supraceliac artery was 100 mmHg, the outlet pressure at each visceral artery was then defined taking into account the corresponding pressure drop for a healthy AA model[First, the entrance boundary of the supraceliac artery was defined as having a constant-velocity inlet of 0.18 AA model. The iliThe graft and vessel wall were assumed to be rigid and nonslip.Meshes in the region of the aorta below the renal arteries, the aortic bifurcation and along the branch vessels were refined. In addition, the boundary layer was resolved by placing the first four grid nodes at approximately 15, 17, 19 and 21μm away from the wall, and we supplemented the zoom-in view of the boundary layer mesh in The flow visualization and analysis were completed by the commercial CFD software Ansys FLUENT 12.0 which was based on the finite volume method. The default segregate implicit 3D solver was applied. Discretization of the equations involved a second order upwind differencing scheme, SIMPLE was adopted for the pressure velocity correction and the residual error convergence threshold was set as 1e-5.To establish a grid-independent solution, a fine mesh consisting of approximately 950914 cells and a coarse mesh of 870319 cells for the unilateral CIA model while two meshes of 273728 and 265250 cells, respectively, for the bilateral CIA model were tested. As is shown in the WSS distribution along the aorta centerline, the maximum relative errors of the WSS magnitudes were 2.3% and 3.5%, respectively . Therefo2 in the arteries[2 for only very brief durations[2, are marked in blue while extremely high WSSs, larger than 100 dyne/cm2, are marked in red in the Figures. There are three prominent features needing to be addressed. First, compared to the bi-furcated graft, the four-furcated graft resulted in more areas with low WSS. For example, most areas of the four-bifurcated visceral grafts were plagued with low WSS while only part of the side walls of the bi-furcated graft were affected. In addition, although for both cases, there were low WSS regions on the artery floor at the anastomosis, only the toe and swing section of the quar-furcated graft suffered from low WSS. Secondly, when a bi-furcated graft was set up, the WSS on its iliac arteries were in the normal range but it was not true when a quar-furcated graft was adopted. Finally, when a unilateral quar-furcated graft was used, extremely high WSS appeared on the aorta bifurcation and anastomosis region of the inflow site , which is not the case when bilateral bi-furcated grafts are deployed. 3) The symmetrical bilateral bi-furcated graft brought symmetrical flow splits to the iliac arteries, but extreme low flow volume to the host iliac artery was observed when a quar-furcated graft used. However, the bilateral bi-furcated grafts resulted in a more significant decrease in flow to the visceral organs, especially a more remarkable decrease in flow to the celiac and superior mesenteric arteries.This paper aims to investigate the general features and phenomena of the hemodynamics in an aorta with RVR for patients with Crawford II/III/IV TAAA. We adopted idealized 3D aorta geometries with a quar-furcated graft from unilateral CIA or bi-furcated grafts separately from bilateral CIA for the numerical investigation. The computational results revealed that: 1) No matter whether a unilateral quar-furcated graft or a bilateral bi-furcated graft, RVRs from the CIAs result in a dramatic decrease in the flow split to the visceral organs, compared with a healthy AA. The unilateral grafts have a 30% decrease while the bilateral grafts have a 43% decrease, which represents a potential risk of inadequate perfusion after the RVR operation. 2) No matter the method of reconstruction from CIAs, the anastomosis regions are plagued with low and unevenly distributed WSS, flow recirculation and stagnation and flow skewness. In addition, comparison between the two different reconstruction from the CIAs showed that the bifurcated grafts from bilateral CIAs have the following benefits: 1) the employment of bilateral bi-furcated grafts for visceral rerouting significantly eliminated the low WSS region, WSS gradient change and flow recirculation, not only in the anastomosis region but in the graft sections, compared to a unilateral quar-furcated graft, 2) When a unilateral quar-furcated graft is set up, parts of its anastomosis region are exposed to extremely high WSS (more than 100 dyne/cm2 is required to keep the normal cell morphology in the human blood vessels. Low WSS is believed to trigger an inflammatory-cell-mediated pathway, while high WSS triggers a mural-cell-mediated pathway, which of both are associated with the growth and rupture of aneurysms[It is well accepted that hemodynamics is one of the most important factors contributing to aneurysm pathophysiology and plays a fundamental role in the mechanisms of initiation, growth, and rupture,22. Espeaneurysms. In addianeurysms. For exaaneurysms.Accordingly, in terms of hemodynamics, we believe that for the Crawford II/III/IV TAAA patients who go through RVR from CIAs, a big concern is the inadequate perfusion to the visceral organs, and regular follow-up monitoring of the function of the visceral organs are highly recommended. In addition, the anastomosis region is highly dangerous for the initiation and the progression of stenosis and aneurysm, and graft design including graft size, and anastomosis angle may be carried out later on to reduce the risk of the related complications of the stent graft or blood vessel prostheses or the potential dilation of CIA. Moreover, when the CIA is the only candidate for the RVR of patients with Crawford II/III/IV TAAA, we believe that a bilateral bi-furcated graft has better performance in suppressing the initiation and development of graft occlusion, and in decreasing the vessel wall dilation and reducing artery aneurysms developing at the host iliac artery which helps to prolong the graft patency.As a preliminary investigation, there are some possible limitations in our investigation:The simulations were carried out under steady flow conditions, which are different from in vivo pulsatile flow conditions. Flow pulsatility may play important protective roles in respect of atherosclerosis and aneurysm, and a large amplitude pulsation may increase the values of the minima in the WSS and velocity, thus enhancing the ‘sweeping’ motion in the blood vessels. But as Wall compliance is neglected in this study, which is not true in vivo; however, previous studies have indicate that the wall elasticity may be of considerable significance in, for example, transport mechanisms, but of somewhat lesser importance as far as the gross features of the flow are concerned.The assumptions of constant viscosity ignores the non-Newtonian property of blood rheology. However, previous studies have indicated that as far as the hemodynamic parameters such as WSS, flow field and pressure drop and so on are concerned, the results predicted by computations using non-Newtonian properties are quantitatively and qualitatively close to those assuming a Newtonian fluid, hence, we found it reasonable to make the assumption of Newtonian blood in this study.Vascular surgeons are more concerned about surgical techniques for TAAA treatment while ignoring different hemodynamic effects from different reconstructions. This study firstly indicated hemodynamic characteristic with RVR from CIAs, and in spite of the limitations mentioned above, revealed the potential risks, including the visceral organ ischemia, graft stenosis, thrombosis and anastomosis aneurysm after an RVR operation, and pointed out the advantages and disadvantages of different bypass procedures. Surgeons will plan a precise perform with patient specific for TAAA or TAAD base on this results. Future clinical or animal studies are needed to explore how the decreased flowrate to the visceral organs may affect their function and confirmed the threshold of visceral or limb ischemia. In addition, at this time, the choice of a bypass graft for RVR is limited and we expect that, in the near future, graft design including graft size and anastomosis angle etc. may help to improve the graft patency and decrease postoperative complications. Most importantly, this study again emphasizes the importance and necessity of regular postoperative follow-up to patients for the better recovery.


Urine α- and π-GST values were measured. Kidney recipients were grouped into patients with acute graft rejection (AGR), calcineurin inhibitor toxicity (CNI), and delayed graft function (DGF), and compared to those with unimpaired graft function. Urinary π-GST revealed significant differences in deceased kidney donor recipients with episodes of AGR or DGF at day one after transplantation . High π-GST values at postoperative day 1 (cutoff: >21.4 ng/mg urine creatinine (uCrea) or >18.3 ng/mg uCrea for AGR or DGF, respectively) distinguished between rejection and no rejection  as well as between DGF and normal-functioning grafts . In living donor recipients, urine levels of α- and π-GST were about 10 times lower than in deceased donor recipients. In deceased donors with impaired graft function in corresponding recipients, urinary α- and π-GST were elevated. α-GST values >33.97 ng/mg uCrea were indicative of AGR with a sensitivity and specificity of 77.7% and 100%, respectively. In deceased donor kidney transplantation, evaluation of urinary α- and π-GST seems to predict different events that deteriorate graft function. To elucidate the potential advantages of such biomarkers, further analysis is warranted.The aim of this study was to analyze the value of urine α- and π-GST in monitoring and predicting kidney graft function following transplantation. In addition, urine samples from corresponding organ donors was analyzed and compared with graft function after organ donation from brain-dead and living donors. Urine samples from brain-dead ( Kidney transplantation is by far the best therapeutic option for patients with end-stage renal disease (ESRD). After transplantation, the main challenges, besides surgical complications, are acute graft rejection, delayed graft function, and adverse effects of immunosuppressants . Acute gThe signs of graft dysfunction are decreased diuresis and impaired creatinine blood levels. Monitoring immunosuppressive drugs and their toxicity through serum levels is of limited value, since the difference between therapeutic and toxic levels is not fixed . In ordeα- and π-GST, which are specifically present in the kidney tubules, are two isotypes of the glutathione-S-transferases. Beyond their biochemical differences, they are also located in different parts of the tubule system . α-GST iThe aim of our study was to determine the value of measuring α- and π-GST concentrations in urine as biomarkers for monitoring graft function and predicting postoperative events in the first week after transplantation in living and deceased donor kidney transplantation.This study was approved by the Ethics Committee of Berlin’s Charité University Hospital (EA2/137/10). We prospectively analyzed blood and urine samples as well as demographic data from 160 patients: 30 brain-dead donors and their 30 corresponding recipients; and 50 living kidney donors as well as their 50 corresponding living donor kidney recipients. All surgeries were carried out at the Department of General, Visceral and Transplantation surgery of Charité University Hospital, Berlin. Machine perfusion was not performed for any kidney allograft in this study. Except for brain-dead donors, all patients were followed during the first week after surgery. Blood and urine samples were collected at the following time points: day 0, day 1, day 3, day 5, and day 7. Samples from brain-dead organ donors were obtained on day 0, the day of the organ donation surgery. Urine samples were collected from recipients after transplantation through an externalized uretero-vesico-cutaneous stent and therefore exclusively reflected the α- and π-GST content of the transplanted grafts.All recipients received a triple immunosuppressant consisting of prednisolone, mycophenolate mofetil (MMF), and a calcineurin inhibitor. All deceased donor kidney recipients received tacrolimus, whereas living donor kidney recipients were treated with either tacrolimus or cyclosporine. Recipients were divided into subgroups according to the events in the first postoperative week: acute graft rejection , calcineurin-induced nephrotoxicity , both acute kidney rejection and calcineurin-induced nephrotoxicity , delayed graft function , and event-free  subgroup . An acutUrinary α- and π-GST values were measured using a commercially available ELISA test kit provided by Argutus Medical Ltd. . To consider the physiological differences in urine concentrations, α- and π-GST values were standardized to urine creatinine. The resulting unit for GST was ng/mg uCrea. Reference ranges of urinary α- and π-GST were determined as recommended from the measured GST values in the healthy population of this study group. For this purpose, the α- and π-GST values in the urine of healthy living donors were considered before donor nephrectomy. The reference interval for α-GST is 2.7–7.6 ng/mg uCrea and for π-GST 4.1–13 ng/mg uCrea in this study.t-tests such as Mann‒Whitney and Wilcoxon were performed. For the comparison of multiple variables, we used two-way ANOVA; here we applied Tukey and Holm‒Sidak tests for post hoc analysis of the subgroups. The area under the curve was calculated in ROC analysis and a log-rank (Mantel‒Cox) test was performed in survival analysis. A p-value less than 0.05 was defined as significant.For statistical analysis, GraphPad Prism 6  was used. Quantitative data are given as the mean and standard deviation. To compare normally distributed variables, p = 0.006), whereas the donor’s age was not different. Cold as well as warm ischemic time were significantly higher in the deceased donor group (both p < 0.0001). The demographic data of donors and recipients are given in There were no differences regarding gender and BMI between the patient groups. The mean eGFR before graft recovery or transplantation (d0) did not differ between the groups. Recipients of deceased donor kidneys were significantly older than those of living donors . In living donor recipients, only π-GST showed an increase, reaching a peak at day 5 (mean 21.5 ± 28.2 ng/mg uCrea); however, there was no significant difference between this group and the healthy subgroup. In patients with CNI toxicity, α-GST performed better in both recipient groups; in deceased donor recipients, the mean was 316 ± 704.5 ng/mg uCrea at 1 POD and was easily discriminated from the uneventful subgroup (p = 0.06). Also, in the living donor group, a rise of α-GST to 68.9 ± 219 ng/mg uCrea was noted at day 5, when CNI toxicity occurred. When both acute rejection and CNI toxicity were recorded, neither α- nor π-GST was able to distinguish those patients; however, it is worth mentioning that the number of subjects in this subgroup was very low in our study. In the case of delayed graft function, present only in deceased donor recipients in our study, α- as well as π-GST were elevated in the urine, with means at 1st POD of 81.5 ± 201.3 and 151.6 ± 270.6 ng/mg uCrea, respectively. Only π-GST levels proved significant when compared to the control subgroup (p = 0.036)  and eight patients (16%), calcineurin-induced nephrotoxicity in five (16.7%) and 12 (24%) patients, and both simultaneously in three (10%) and three (6%) patients, respectively. Delayed graft function occurred only with deceased donors . Patients with an uneventful postoperative course in the deceased donor recipient group numbered nine (30%) and, in the living donor recipient group, 27 (54%). Both α- and π-GST were significantly elevated at 1st postoperative day (POD) in deceased donor recipients, with acute rejection when compared with the corresponding healthy subgroup  .Furthermore, we performed ROC curve and survival analysis on the most outstanding biomarkers at a specific point in the study. π-GST showed the most promising results in deceased donor recipients with acute graft rejection and delayed graft function at day 1 after transplantation. Patients who developed a biopsy confirmed acute graft rejection within the first week after transplantation had significantly higher levels of urinary π-GST at POD 1. With an estimated cutoff of 21.4 ng/mg uCrea, π-GST was able to distinguish the occurrence of AGR from a rejection-free course with 100% and 66.6% sensitivity and specificity, respectively . Similarp = 0.02), while α- and π-GST were remarkably higher (but not significantly so) in the DGF subgroup compared to the control group (p = 0.0109)  .On the other hand, urinary GST in living donors showed no differences between subgroups and the corresponding control group, and therefore failed to predict future events in recipients.We followed up recipients of the subgroups G1 (AGR) and G4 (DGF) at six and 12 months after transplantation, as correlations of α- and π-GST in those cohorts showed the most promising results. However, in subgroup G1 only one patient out of four lost the graft due to recruiting nephritis; in subgroup G4 all three grafts were lost due to death not associated with graft function (cancer or cardiac arrest).The results of our prospective study, evaluating urinary α- and π-GST in deceased as well as living kidney donors and their corresponding recipients as biomarkers for graft quality and function, suggest the potential value of these enzymes. Previous studies showed the ability of urinary α- and π-GST to predict acute renal damage in kidney graft recipients and demonstrated a release of these enzymes in malfunctioning grafts ,13,14,15Research studies investigating α- and π-GST in living donation are extremely limited: in our review of the literature we only found one publication on this issue, and this concerned liver rather than kidney transplantation . Our finThe toxic effect of CNI agents on renal grafts and its association with the excretion of α-GST has been described in the past ,18. Our DGF was observed only in deceased donor recipients in our study. It occurred with an incidence of 30%, which is similar to the findings of a recent work by Willicombe et al. . Risk faFurther limitations of this study are the small number of patients in certain subgroups and the overall high standard deviations. We distinguished well between the causes of impaired graft function and took into consideration simultaneous events. The time frame of our observation was limited in that it focused only on the first week after transplantation. We believe that multiple serial samples and an extended study design would be beneficial in future projects.In summary, the elevation of urinary π-GST in deceased donor kidney recipients at day 1 after kidney transplantation could be a helpful monitoring parameter, in addition to urinary output and serum creatinine, to determine graft function in recipients. It might be indicative of acute rejection or a need for dialysis. The measuring of urinary α- and π-GST should also be considered in deceased donors as this seems to be of predictive value in terms of graft outcome and might help with assessing allograft quality. Our findings reveal an association between urinary α-GST in deceased donors and AGR in corresponding recipients. Thus, further investigation of α- and π-GST in a larger population and daily sample collection should be considered. Although urinary α- and π-GST in living kidney donation showed no relevant correlation with harmful events in our analyses, this is, to the best of our knowledge, the first study demonstrating differences in biomarkers between deceased and living kidney donation, so subsequent investigations will be needed in order to confirm or contradict our findings.


Our understanding of the anatomy of hearts of ectothermic saurosids, or colloquially “reptiles”, was much advanced by the publication of the series of four papers under the heading of Comparative Cardiac Anatomy of the Reptilia in Journal of Morphology between 1971 and 1981. Here, I commemorate the papers, show how they moved our understanding forwards, and briefly describe the state‐of‐the‐art. Fifty years ago, in Sydney Australia, Grahame Webb finished his thesis “The squamate heart” that would become the foundation of the first two of ultimately four papers of the classical series “Comparative Cardiac Anatomy of the Reptilia” published in Journal of Morphology , but not the vertical septum of a lizard (Anolis). Later, Poelmann et al.  and a snake (Pantherophis) that the crest of the vertical septum is enriched in Tbx5 and that there is an additional gradient of Tbx5 on the Muskelleiste . While the newer findings questioned whether a gradient of Tbx5 is a marker of any particular septum, Jensen et al.  and archosaurs (Sauropsida). Investigations showing a gradient of the transcription factor In the inaugural issue of Journal of Morphology, the editor Charles Otis Whitman envisioned the journal as a “medium” to concentrate and remedy the “mixed character and scattered sources of our publications” Whitman, . Journal