Healthy diet has been shown to alter brain structure and function and improve cognitive performance, and prior work from our group showed that Mediterranean diet (MeDi) moderates the effect of between-network resting-state functional connectivity (rsFC) on cognitive function in a cross-sectional sample of healthy adults. The current study aimed to expand on this previous work by testing whether MeDi moderates the effects of changes in between- and within-network rsFC on changes in cognitive performance over an average of 5 years.At baseline and 5-year follow up, 124 adults aged 20–80 years underwent resting state fMRI to measure connectivity within and between 10 pre-defined networks, and completed six cognitive tasks to measure each of four cognitive reference abilities (RAs): fluid reasoning (FLUID), episodic memory, processing speed and attention, and vocabulary. Participants were categorized into low, moderate, and high MeDi groups based on food frequency questionnaires (FFQs). Multivariable linear regressions were used to test relationships between MeDi, change in within- and between-network rsFC, and change in cognitive function.Results showed that MeDi group significantly moderated the effects of change in overall between-network and within-network rsFC on change in memory performance. Exploratory analyses on individual networks revealed that interactions between MeDi and between-network rsFC were significant for nearly all individual networks, whereas the moderating effect of MeDi on the relationship between within-network rsFC change and memory change was limited to a subset of specific functional networks.These findings suggest healthy diet may protect cognitive function by attenuating the negative effects of changes in connectivity over time. Further research is warranted to understand the mechanisms by which MeDi exerts its neuroprotective effects over the lifespan. A growing body of research suggests modifiable lifestyle factors, including healthy diet, may protect cognitive function against negative effects of age- and disease-related changes in brain structure and function. Several healthy dietary patterns, as well as individual food types and nutrients comprising healthy dietary patterns, have been shown to benefit brain and cognitive health . One of The biological mechanisms by which healthy diet contributes to cognitive function is not well understood, but some studies have suggested components of a healthy diet may alter cytokine activity and apoptotic pathways to reduce inflammation and oxidative stress , and heavia experience-driven changes in connectivity, wherein repeated co-activation among regions engaged during cognitive tasks leads to changes in functional networks at rest  among large-scale networks at rest tends to increase with age, reflecting reduced segregation and specialization of networks underlying cognitive functions . Convers at rest , and inc at rest  and long at rest  studies, at rest .In the first study to date to investigate the relationship between MeDi, functional connectivity, and cognitive performance, work from our group recently showed that MeDi adherence moderated the cross-sectional relationship between rsFC and cognitive performance: for individuals less adherent to MeDi, lower between-network rsFC was associated with poorer fluid reasoning (FLUID) performance, but the relationship between rsFC and cognition was attenuated in those with moderate and high MeDi adherence . These fThe present study aimed to (1) test the relationship between MeDi and longitudinal change of between- and within-network rsFC, and (2) test whether MeDi moderates the longitudinal association between rsFC and cognitive function in a sample of healthy adults across the lifespan. Because diet has been shown to impact whole-brain measures of brain structure and function , as wellN = 25), or baseline and follow-up rsFC data (N = 55); of the 174 remaining participants, 49 were excluded due to missing diffusion tensor imaging fractional anisotropy (DTI FA) data, and 1 due to missing white matter hyperintensity (WMH) data, resulting in a final sample size of N = 124.The current study included adults aged 20–80 years, who completed the baseline and follow-up visits for two ongoing studies: the Reference Ability Neural Network (RANN) study  and the All participants were native English speakers, right-handed, free of MRI contraindications, and read at a fourth-grade level or above. No participants had any psychological or medical conditions that could affect cognitive function, and no older adults met criteria for dementia or MCI. All participants provided informed consent, and all methods were approved by and performed in accordance with the relevant guidelines and regulations of the Institutional Review Board of the College of Physicians and Surgeons of Columbia University.Habitual dietary information in the past year was self-reported at the baseline visit using a 61-item version of Willett’s semi-quantitative food frequency questionnaire (FFQ) . FrequenCognitive function was measured using performance on six cognitive tasks for each of four reference abilities (RAs): FLUID, processing speed and attention (SPEED), memory (MEMORY), and vocabulary (VOCAB). As described in previous work from our group , each abMagnetic resonance images were acquired on a 3T Philips Achieva Magnet, over two 2-h sessions. Participants underwent 9.5 min of fMRI blood oxygen level-dependent (BOLD) resting state scans. T1-weighted whole brain images were acquired for each subject using magnetization-prepared rapid gradient-echo (MPRAGE) sequence. Diffusion tensor imaging (DTI) and fluid-attenuated inversion recovery (FLAIR) scans were also acquired. Scanning parameters can be found in Resting-state fMRI pre-processing included discarding the first three volumes, slice-timing correction and motion correction performed in FSL , frame-wDiffusion tensor imaging data were pre-processed through standard pre-processing pipeline including eddy- and motion correction using FSL. TRACULA  toolbox,via inverse transformation based on the non-linear registration of each subject’s structural scan to MNI template using ANTS software package. Ten mm radius spherical masks were generated for each coordinate and intersected with the FreeSurfer gray matter mask to obtain the gray matter-registered ROI masks for each ROI. An intermodal, intra-subject, rigid-body registration of the fMRI reference image and T1 scan was then performed using FLIRT with 6 degrees of freedom, normalized mutual information as the cost function ; middle-aged adult ; older adult ].For between-network rsFC, exploratory analyses were then conducted to examine the 10 individual network-based connectivity, i.e., the mean correlation between ROIs in each network and ROIs in the nine other networks, in order to probe network-specific effects, producing 10 values reflecting mean between-network connectivity from each of the 10 networks. Similarly, for within-network rsFC, exploratory analyses were conducted to examine connectivity among all ROIs within each of the networks, producing 10 values reflecting mean connectivity within each network. Further exploratory analyses for both between-network and within-network rsFC included stratifying the above moderation analyses by age groups based on tertiles: , years of education , NART IQ , cortical thickness , total brain volume , WMH , DTI FA , or baseline total scrub percentage . There was a significant difference in caloric intake among MeDi groups , with greater caloric intake in the high MeDi group. Chi-squared tests showed MeDi groups did not differ by race , but females were more likely to have middle and high tertile of MeDi than males .One-way analyses of variance showed there were no significant differences between MeDi groups in baseline measures of mean age  change in overall within-network rsFC , or change in performance on the four RAs: FLUID , MEMORY , VOCAB , SPEED  , wherein the moderate MeDi group had greater increase in overall between-network rsFC than the high MeDi group . MeDi group was not associated with overall within-network rsFC change (p = 0.275). MeDi group was not a significant predictor of change in MEMORY (p = 0.366), FLUID (p = 0.956), SPEED (p = 0.264), or VOCAB (p = 0.128) performance from baseline to follow-up visits.Analyses using multivariable linear regressions controlling for covariates described above revealed a marginally significant effect of MeDi group on overall between-network rsFC change among all 10 networks (p-interaction = 0.033): relative to the low MeDi group, the association of the between-network rsFC change with the change in MEMORY was weaker in the moderate MeDi group (p-interaction = 0.053) and high MeDi group (p-interaction = 0.010) , with no significant effect of change in between-network rsFC on MEMORY change for the moderate MeDi group. For the high MeDi group, there was a significant positive effect of change in between-network rsFC on change in MEMORY  (p-interaction = 0.713), SPEED (p-interaction = 0.748), or VOCAB (p-interaction = 0.398)  , suggest= 0.009) . MeDi di= 0.398) .p-interaction = 0.028): relative to the low MeDi group, the association between within-network rsFC change and change in MEMORY was weaker in the moderate MeDi group (p-interaction = 0.039) and high MeDi group (p-interaction = 0.011) , no significant relationship in the moderate MeDi group, and a significant positive effect of change in rsFC on change in MEMORY in the high MeDi group . MeDi did not moderate the effect of change in overall within-network rsFC on change in FLUID (p-interaction = 0.811), SPEED (p-interaction = 0.896), or VOCAB (p-interaction = 0.663) performance.For MEMORY, we found a significant interaction between MeDi group and overall within-network rsFC change on change in performance (= 0.011) . Analysep = 0.023), Auditory (p = 0.022), CO (p = 0.054), DAN (p = 0.098), and Salience (p = 0.011) networks  and between change in overall within-network rsFC on change in memory , with no significant effects for other cognitive domains .Sensitivity analyses were also conducted to examine whether MeDi moderated associations between changes in overall rsFC and changes in cognition when MeDi scores were modeled as a continuous rather than categorical variable. Results showed that continuous MeDi score remained a marginally significant moderator of the relationship between change in overall between-network rsFC on change in memory (In the current longitudinal population-based study, we found that adherence to MeDi significantly moderated the relationship between change in both between- and within-network rsFC and change in memory performance over an average of 5 years. To our knowledge, this is the first study to test the relationship among MeDi, change in connectivity at rest, and change in cognitive performance, and demonstrate that MeDi may benefit cognition by mitigating the negative effect of altered connectivity on cognitive function over time.Of the four RAs tested, MeDi had the most robust effect on the relationship between rsFC and memory. This is consistent with past research suggesting episodic memory is among the cognitive domains most influenced by effects of both normal aging and neuropathological change . The finA considerable amount of past research has also suggested that within-network rsFC tends to decrease with age, and that these decreases in connectivity often predict decreases in cognitive function . In contThe mechanisms by which MeDi protects against negative cognitive effects of neurological change remain unclear, but past research has suggested foods comprising MeDi may reduce inflammation and oxidative stress . MeDi maTo our knowledge, this is the first longitudinal study to examine the effects of dietary patterns on changes in within- and between-network rsFC, and to evaluate the moderating effect of MeDi on the relationship between changes in rsFC and change in cognitive performance over time. One strength of the current study is the inclusion of adults across a broad age range, in contrast to past literature that has primarily focused on the effects of lifestyle factors on brain and cognitive function in only older adult populations. Our exploratory analyses revealed that older adults showed a greater protective effect of MeDi on the negative cognitive effects of changes in rsFC, suggesting the impact of MeDi may be most beneficial in mitigating age-related neurocognitive decline. The current study is also strengthened by adjusting for the potential confounding effects of structural brain measures on change in rsFC and cognition, which allows us to interpret a direct role of diet on the relationship between connectivity and performance above and beyond structural brain health.The small amount of existing research on the role of diet in neurocognitive change has primarily focused on the effects of individual nutrients, the interpretation of which is limited by the fact that typical diets are composed of multiple potentially interacting and inter-correlated nutrients . MoreoveOne potential limitation of the current study is the use of composite cognitive ability scores based on both in-scanner tasks and out-of-scanner neuropsychological assessments. As such, our results may not directly support or contradict findings in prior research on the relationships between rsFC, MeDi, and specific cognitive processes, but rather, provide important evidence that MeDi moderates the relationship between rsFC changes and changes in broad cognitive domains. Due to limited sample size, we also did not correct for multiple comparisons in our statistical analyses, resulting in the potential for type 1 errors. However, the consistency of our results across nearly all networks of interest suggests a lower likelihood of false-positive results, and these findings are also consistent with our past work showing MeDi moderates the relationship between rsFC and cognitive function in a cross-sectional sample . As suchAnother potential limitation is the use of external parcelation scheme to define resting state networks. Although it is possible to base network parcelation on participants’ own network structures, the wide age range of participants in the current sample could have introduced age-related biases in network assignment. Moreover, previous work from our group and others, including our recent cross-sectional study of the interaction between MeDi and between-network rsFC on cognition , has utiResults of the current study suggest that MeDi may support cognitive function despite changes in brain connectivity, as evidenced by an attenuated effect of change in connectivity on change in cognitive function in those with higher MeDi adherence. Future research is warranted to understand the mechanisms by which MeDi exerts its neuroprotective effects, as well as the role of other modifiable lifestyle factors in the relationship between brain biomarkers and cognitive function over the lifespan.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board of the College of Physicians and Surgeons of Columbia University. The patients/participants provided their written informed consent to participate in this study.YGu and AG: conception and design of the work and drafting of the manuscript. All authors: critical revision and final approval of the manuscript. All authors fulfill the ICMJE criteria for authorship.


Small-molecule contaminants, such as antibiotics, pesticides, and plasticizers, have emerged as one of the substances most detrimental to human health and the environment. Therefore, it is crucial to develop low-cost, user-friendly, and portable biosensors capable of rapidly detecting these contaminants. Antibodies have traditionally been used as biorecognition elements. However, aptamers have recently been applied as biorecognition elements in aptamer-based biosensors, also known as aptasensors. The systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro technique used to generate aptamers that bind their targets with high affinity and specificity. Over the past decade, a modified SELEX method known as Capture-SELEX has been widely used to generate DNA or RNA aptamers that bind small molecules. In this review, we summarize the recent strategies used for Capture-SELEX, describe the methods commonly used for detecting and characterizing small-molecule–aptamer interactions, and discuss the development of aptamer-based biosensors for various applications. We also discuss the challenges of the Capture-SELEX platform and biosensor development and the possibilities for their future application. Accumulating evidence suggests that small-molecule contaminants, such as antibiotics, pesticides, and plasticizers, pose a severe threat to human health and the environment worldwide. Antibiotics have been extensively used for decades to treat bacterial infections, and most are recalcitrant to biodegradation and elimination . MoreoveSome traditional techniques for detecting small-molecule contaminants with high accuracy include high-performance liquid chromatography ,8, enzymAptamers are short, single-stranded DNA or RNA oligonucleotides that are able to bind its target with high affinity and specificity. The development of an aptasensor for detecting small-molecule contaminants requires the generation of a novel aptamer. It has gradually replaced traditional antibodies in some applications as they have several advantages over antibodies, such as thermal stability, low cost, amenability to chemical modification, and ease of generation ,14. FurtThree research groups independently developed the first SELEX method and all reported their findings in 1990: Tuerk and Gold , EllingtThe classical procedure of the DNA Capture-SELEX selection method involves hybridization, immobilization, target-bound sequence elution, polymerase chain reaction (PCR) amplification, regeneration of single-stranded DNA (ssDNA), and sequencing A. Briefl20 extension or a forward primer labeled with either fluorescein or the cyanine dye Cy3. The gel purification method involves denaturing polyacrylamide gel electrophoresis (PAGE), followed by extraction and purification. The MB-based method begins with PCR products with biotinylated reverse primers being incubated to facilitate their deposition onto MBs. The supernatant is then removed, and the non-biotinylated forward strands of dsDNA PCR products are released from the MBs by treatment with sodium hydroxide and then neutralized by treatment with an appropriate amount of tris(hydroxymethyl)aminomethane hydrochloride or hydrochloric acid. The regenerated ssDNA in each round is collected for the next round of selection and then prepared for sequencing after several rounds of selection. Currently, Capture-SELEX is mostly applied for the selection of DNA aptamers rather than RNA aptamers. RNA Capture-SELEX adopts the same selection strategies as DNA Capture-SELEX, except for including two more steps: transcription and reverse transcription . Sanger sequencing is a DNA sequencing technology based on the chain termination method invented by Frederick Sanger and his colleagues  in 1997.https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 12 October 2022)) ..2- and πThe AuNP colorimetric assay is widely used for characterizing the binding affinity of an aptamer for its target ,80,83,84Kd of an aptamer for its small-molecule target.The MST assay is a powerful tool for testing the binding affinity of aptamers for their small-molecule targets as it only requires a small amount of sample (up to 4  15 min) ,76,80. I2 structure and numerous oxygen-containing functional groups [Kd value of the aptamer for its small-molecule target is calculated [GO can be synthesized from graphite via the Hummers and Offeman method , which ul groups , such as bonding E. When 5lculated ,77,109.The ITC assay is a label-free and immobilization-free assay for measuring the binding affinity of an aptamer for its small-molecule target ,83 in anTo date, Capture-SELEX has been used for screening and generating a series of aptamers against small-molecule contaminants . As aptaOptical-fiber-based BLI biosensors have been developed for real-time, sensitive, and rapid measurement  of interaccuracy .LFAs have been developed as portable detection devices with cost-effective, rapid (≤15 min), and easy operation and broad applications, such as in the detection of pregnancy, severe acute respiratory syndrome coronavirus 2 ,119, andRactopamine (RAC) is a ivestock ,126. Theivestock . Therefoivestock  obtaineductivity . Comparemination . Due to vomiting . Yang etvomiting  used CapVanillin is the second most popular flavoring agent worldwide and is used as a food additive in sweet foods and beverages, and a masking agent in numerous pharmaceutical formulations . It is aAnother aroma compound, furaneol, is extensively used as an artificial flavoring agent as it imparts fruit flavor to food , and thuμM .m) in the screening process, and Tm peaked during the 14th round for both TYR and PHE. The aptamers TYR-2 and PHE-2 were identified to have the strongest binding affinity and specificity for TYR and PHE, respectively, based on a GO-based fluorescent assay. TYR-2 and PHE-2 were then used to develop fluorescent aptasensors for the detection of TYR and PHE in pork and bear meat samples. These aptasensors demonstrated LODs for TYR and PHE of 2.48 and 3.22 nM, respectively , and in foods ,136. Tiain foods  obtainedectively , with taErythromycin is a broad-spectrum macrolide antibiotic used to treat diseases such as diphtheria, pertussis, and bacillary angiomatosis . The natRoxithromycin is a macrolide antibiotic that poses a similar risk to the ecosystem and human health as erythromycin, indicating the need to establish a rapid and effective detection device for monitoring roxithromycin residues in environmental media. Jiang et al. selected aptamer candidates against roxithromycin after 16 selection rounds of Capture-SELEX. The aptamer Ap01 demonstrated the highest affinity and specificity for roxithromycin, as indicated by the results of an SGI assay, and was therefore selected for the development of a colorimetric aptasensor for roxithromycin. The developed aptasensor demonstrated a low LOD (0.077 μM) for roxithromycin in water samples  and highKd: 288 nM) and was therefore chosen for further optimization and investigation. The truncated aptamer G48nop demonstrated an improved Kd value of 34.5 ± 1.72 nM and high specificity for GYM-A. A novel BLI-based aptasensor was established using this aptamer that detected GYM-A in the range of 55–1400 nM (linear range of 55–875 nM) and had a low LOD  are fast-acting cyclic imine toxins that are biosynthesized by dinoflagellates and have deleterious effects on the aquatic environment with the accumulation. The contaminated environment can have serious toxic effects on filter feeding shellfish and thereby pose a threat to human health . Zhang e6.21 nM; . This BLDi(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used as an additive in packaging materials, and its residues are known to accumulate and dissolve in water . DEHP isKd: 21 nM) using an ITC assay. Through further in vivo selection, this aptamer was revealed to have riboswitching properties. This work has introduced an efficient protocol for developing synthetic riboswitches and boosted the development of real-time intracellular biosensors for monitoring metabolic flows in living cells.Synthetic riboswitches can have several biotechnological applications, such as regulating gene expression, e.g., the construction of genetic circuits ,142. NatZearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by fungi and is known to contaminate cereal grains and other crops . Due to Fenitrothion (FEN) is a broad-spectrum organophosphorus insecticide mainly used to control insect pests in agriculture . As FEN Small-molecule contaminants are ubiquitous in the aquatic environment, agriculture produce, and animal feed due to the overuse of small molecules such as antibiotics and pesticides, and these contaminants pose a serious threat to human health and the environment. Owing to the few functional groups on these small molecules, screening aptamers against them is more challenging than doing so against large-molecule targets. Compared with other SELEX approaches, which involve the immobilization of small-molecule targets, the Capture-SELEX approach is more feasible, as they involve the immobilization of a biotinylated ssDNA/RNA library against which the binding affinity and specificity of small-molecule targets can be screened. To date, fewer than 50 studies have reported using Capture-SELEX to identify novel aptamers against specific small-molecule contaminants, suggesting that this process remains challenging, such as false-positive and lack of diversity. However, this research field has recently been receiving increasing attention from scientists. We hope that this review will encourage further research into the use of Capture-SELEX in generating aptamers against small-molecule contaminants. Six small-molecule aptamer characterization methods are introduced in this review. The high affinity and specificity aptamer work as a biorecognition element in aptasensor to detect specific small-molecule contaminants in environmental media and agricultural produce. This will help to improve food safety, aquatic environments, and agricultural crop production.


Few studies have directly compared the clinical feasibility of electrocardiography-gated and non-electrocardiography-gated multidetector computed tomography for evaluating coronary arteries in veterinary medicine. We aimed to characterize and visualize feline coronary arteries using these two imaging modalities. We hypothesed that ECG-gated MDCT is superior to or advantageous to the non-ECG gated.This prospective, controlled, comparative pilot study examined six client-owned cats  using non-electrocardiography-gated and retrospective electrocardiography-gated scans. Optimal non-electrocardiography scan timing or electrocardiography-gated R-R reconstruction interval for coronary artery visualization was determined. The degree of opacification and sharpness of proximal coronary branches was subjectively graded; coronary dominance, left coronary artery branching types, and the diameter and length of coronary artery branches were also assessed.Non-electrocardiography-gated images provided the least information on the bilateral coronary artery ostium and proximal segments, while electrocardiography-gated images clarified the detailed course of the main coronary branches at diastole in all cats. The degree of opacification and sharpness of the coronary arteries was subjectively evaluated as good/excellent in all cats. Coronary dominance  and left coronary artery branching types  varied. Body weight and sex were not significantly associated with coronary artery length or diameter. Vertebral heart score positively correlated with the right coronary artery and negatively correlated with the left main coronary artery.Electrocardiography-gated multidetector computed tomography provides images with adequate resolution to identify the anatomy of feline coronary arteries. Detailed morphological knowledge of feline coronary vessels will enable novel diagnostic and therapeutic methods in veterinary medicine. Multidetector computed tomographic (MDCT) angiography is considered the gold standard for evaluating coronary vessels in humans, permitting multiple interventional cardiologic procedures –4. AlthoElectrocardiography (ECG)-gated MDCT links image acquisition to specific phases of the cardiac cycle . In prosPrevious reports on canines indicated that ECG-gated MDCT was more effective in identifying anomalous CAs than non-ECG-gated MDCT, in terms of imaging the cardiac morphology and the detailed CA courses, due to minimizing cardiac motion artifacts , 11–13. Six domestic short-haired cats with no clinical signs provided by the owners were included. Before the MDCT examination, all cats underwent basic health tests, including physical examination, complete blood counts, serum biochemistry, and electrolyte tests. N-terminal Pro-B-type natriuretic peptide (NT-proBNP) testing, thoracic radiography, and transthoracic echocardiography  were performed for cardiac evaluation. Using thoracic radiography, the vertebral heart score (VHS) was calculated. Two-dimensional, M-mode, and Doppler ECG examinations were performed on all cats. The time interval between all basic health tests and MDCT examinations for individual cats was within five days.This study was a prospective, controlled, comparative pilot study including cats that had been brought to an animal medical center for a complete medical check-up. The study design and care, as well as animal maintenance, followed protocols approved by the institutional animal care and use committee of Seoul National University . Medical history and informed consent were obtained for all client-owned cats prior to study procedures. via an endotracheal tube. End-tidal carbon dioxide levels were maintained between 35 and 45 mmHg using a mechanical ventilator. Heart rate, oxygen saturation, and end-tidal carbon dioxide were continuously monitored during anesthesia via ECG and pulse oximetry. Data acquisition was initiated within 5–10 min after anesthesia induction to ensure the stability of anesthetic conditions. For individual scans, apnea was induced by breath-holding at inspiration immediately before the scan. All cats were monitored until recovery from anesthesia. The total time from the start to the end of anesthesia was recorded.An intravenous 24-G catheter was placed in the right cephalic vein for premedication and contrast agent injection during MDCT. The cats were premedicated with butorphanol , and general anesthesia induced with propofol , and maintained with isoflurane  in a gas mixture of 100% oxygen in the air MDCT CA angiography was performed using an 80-row, 160-multislice CT system . For both non-ECG-gated and ECG-gated MDCT, cats were positioned in sternal recumbency on a CT table with the neck extended and the forelimbs positioned caudally. ECG leads were attached to the paws; ECG data were recorded simultaneously during spiral MDCT examination. Scan parameters were as follows: voltage, 120 kVp (kVp: kilovoltage peak); gantry speed, 0.5 s/rotation; slice collimation, 0.5 mm × 80; 150 mA; 0.5 mm slice thickness; and pitch factor, 0.813. All cats were examined using non-ECG-gated scans, followed by ECG-gated scans after a short interval (5 min) to wash out the contrast medium from the heart. In all cats, a pre-contrast MDCT scan of the full thorax from the thoracic inlet to the caudal-most border of the lungs was performed prior to the post-contrast studies. For non-ECG-gated scans, a biphasic injection was administered, comprising a non-ionic contrast medium , followed by a saline flush (1.5 mL/kg) into the cephalic vein using a dual power injector  at a rate of 1.5 mL/s. Image acquisition began 8 s after the initiation of the contrast injection. Subsequently, five sequential scans, cranial to caudal and vice versa, from the second rib to the cranial border of the diaphragm, were performed at 5-s intervals. To reduce radiation exposure and anesthesia time, the delay time for retrospective ECG-gated scans was determined based on non-ECG-gated sequential images. Contrast medium administration for ECG-gated MDCT was conducted in the same manner as that for non-ECG-gated scans. For data postprocessing, images were reconstructed in multiple datasets, with the temporal reconstruction window increasing in 10% increments within the cardiac cycle, centered over the 0–90% R-R interval. All images were reviewed by three veterinary diagnostic imaging experts on a dedicated viewing station using specialized software . All images obtained in the non-ECG-gated and ECG-gated MDCT examinations were evaluated, and the scan timing and phase with the least motion were selected for image reconstruction in standard tomographic views. Maximum intensity projection, three-dimensional volume-rendered, multiplanar, and curved reconstructions were applied as needed to optimize CA visualization .The non-ECG and ECG-gated images were assessed based on the following:(a) Optimal scan timing or phase in the R-R reconstruction interval for the best visualization of the proximal CA branches (from the start point to ~3–5 mm).(b) The degree of opacification and sharpness of the proximal CA branches, which were subjectively graded as poor (0), mild (1), good (2), or excellent (3) . The pre(c) Coronary dominance , which was defined as a left CA (LCA) or right CA (RCA) extending beyond the crux cordis  and the origin of the subsinosal interventricular branch , 17.five main types of the LCA proximal segment, as previously described ((d) Classification of LCA branching into escribed  9). Typ. Typfive(e) The diameter and length of the LCA and RCA according to the segmentation of their branches, based on a method used in previous canine studies , 16. TheP-values were calculated for each contrast. The mean values of sex-related parameters were compared using a two-tailed unpaired Student's t-test. P-values < 0.05 were considered statistically significant.All data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0.2 software . Data were assessed for conformance to a normal distribution using the Shapiro-Wilk test. Linear correlations of body weight (BW), sex, and VHS with the diameter and length of the major coronary branches were identified using Pearson's chi-squared test. Pearson's correlation coefficients, 95% confidence intervals (CIs), and Six cats  comprised three spayed females and three males (one intact and two neutered) . Five caMDCT angiography using non-ECG-gated and ECG-gated scans was performed successfully on all cats. The average total time from the induction of anesthesia to the conclusion of MDCT examination was 30 min  per cat. Heart rates during the MDCT scan were 120–150 bpm in all cats. No complications associated with the anesthetic protocol were documented in any cat. The delay time for the retrospective ECG-gated scan, determined using the second scan timing images for non-ECG-gated scanning, was set at 14 s in all cats.In non-ECG-gated images, the first scan exhibited no contrast enhancement in the left heart, which precluded the evaluation of the left heart and CAs in all cats . The secIn ECG-gated images, the optimal R-R reconstruction intervals for both the LCA and RCA were 70, 80, and 90% in two, one, and two cases, respectively . In lineIn selected images, the degree of opacification and sharpness of the proximal coronary branches was subjectively evaluated as excellent  or good  . ECG-gatFour cats exhibited left dominance; two exhibited right dominance .CA branching types were characterized as type II in four cats, type I in one cat, and type III in one cat . In the Left and right CA branches were further classified by adapting a previously described segmental coding system, whereby an arterial portion located between two reference points was considered an angiographic segment  16). Th. Th16). two cats, ran laterally to the aortic bulb toward the interventricular septum (S1) and turned perpendicular to the scanning plane just ventral to the aortic bulb supplying the mid-caudal aspect of the interventricular septum (S2).The three Cx segments were termed Cx1 , Cx2 , and Cx3 . The cranial segment ran caudally and ventrally to the left auricle and continued caudolaterally along the lateral surface of the left ventricular myocardium. The lateral segment ran perpendicular to the scanning plane into the coronary groove, laterally to the left atrioventricular junction, and ventral to the left circumflex vein. The caudal segment ran caudomedially to the left atrium, parallel to the scanning plane and along the caudodorsal surface of the left ventricle, and continued to run in the subsinosus interventricular groove, ending as the subsinosus interventricular branch. The Pc was classified into three segments: dorsal (Pc1), medium (Pc2), and apical (Pc3). The dorsal segment ran along the mid-dorsal aspect of the heart, caudoventrolaterally to the left, just caudal to the main pulmonary artery, ventromedially to the left auricle, and almost parallel to the scanning plane. The medium segment continued to run caudally along the mid-surface of the heart and within the Pc interventricular groove before ending as Pc3 along the ventral portion of the heart . The S branch, which was observed only in The RCA originated from the right sinus of Valsalva and was classified into three segments: RCA1, RCA2, and RCA3. The first segment ran ventrally in the coronary groove, between the main pulmonic trunk and right atrium, almost parallel to the scanning plane. The second segment turned perpendicular to the scanning plane ventrolateral to the right atrium, into the right atrioventricular groove. The third segment ran caudodorsally, almost perpendicular to the scanning plane, and continued into the right atrioventricular groove on the diaphragmatic surface of the heart.P-values of the analyses for all major CA branches, BW, sex, and VHS are also provided (P > 0.05) (P < 0.05)  . We obse < 0.05) .The current findings demonstrate that ECG-gated MDCT angiography is an effective method for obtaining diagnostic images for the evaluation of feline CAs, without the need for heart rate modulation or beta-blocker administration. The scanning protocol used herein is the first to set the delay time for ECG-gated scans to non-ECG-gated images without bolus tracking or the use of a test bolus, thereby permitting the successful acquisition of ECG-gated scans. Although the scanning method was performed with the aim of directly comparing non-ECG and ECG-gated images in the same patient, our findings indicate that selecting the delay time, reducing radiation exposure, and widening the fourth or fifth non-ECG scan range may be beneficial for patients that require abdominal or whole-body scans beyond the heart. Therefore, the scanning method used herein may be clinically useful in veterinary medicine.The detailed course of the CAs was difficult to evaluate on non-ECG MDCT scans due to poor opacification and severe motion artifacts, although the bilateral coronary ostium and proximal CA segments could be visualized on the second and third scans. These findings indicate that non-ECG MDCT scans can provide the minimal information required to evaluate congenital single CA diseases, similar to findings observed in canines , 11, 13.This study is the first to describe feline LCA branching types using cardiac MDCT. Type II, which does not involve S branches, was the most predominant type, occurring in four of the six cats. This type has not been reported in dogs to date , 16, 18.In the current study, the diameters of the LCA and RCA were considered short relative to their length. The diameters of the LMCA and RCA were 1.42 ± 0.23 and 1.3 ± 0.17, respectively, in contrast to a previous report indicating that the diameter of the RCA was slightly larger . We alsoCoronary dominance of the heart refers to whether the LCA or RCA perfuses the majority of the myocardial tissue. Variable methods have been adopted to determine this parameter , 17. ForIn the current study, the cat diagnosed with HCM during a basic health check-up exhibited a positive result on pro-BNP tests, cardiomegaly (VHS 8.2 v) on thoracic radiography, and regional thickening of the interventricular septum (0.66 cm) in the right parasternal short-axis view on ECG. As there were no abnormalities in the left atrium-to-aorta ratio , diastolic function, systolic anterior motion of the mitral valve, or other examinations, the cat was able to undergo a cardiac CT scan. The cat with HCM exhibited a type II LCA branching pattern, which was the most predominant in our study, as well as right coronary dominance and marked right coronary length. This may have partly underpinned the positive correlation between VHS and RCA herein. HCM is a primary myocardial disorder, indicating that myocardial abnormalities are due to a defect, most often within the sarcomeres  of the cardiomyocytes, and are not secondary to other causes, such as hyperthyroidism, systemic hypertension, aortic stenosis, or acromegaly . We hypoThis study has some limitations, including a small sample size that comprised only five healthy cats and one with HCM. Further, we did not obtain confirmation based on gross findings or biopsy. Despite these limitations, our prospective study of feline CAs using MDCT holds clinical significance for veterinary medicine. In human medicine, the assessment of CA morphology is performed not only to diagnose certain diseases but also before open and endovascular or cardiovascular procedures . FurtherIn conclusion, the cardiac MDCT scanning protocol, comprising non-ECG followed by ECG-gated MDCT scans, enabled adequate visualization of CAs without bolus tracking and provided useful information concerning feline CAs. These findings suggest that cats may potentially exhibit varieties of normal CA patterns. Further studies in a big number of healthy and cardiomyopathic cats are warranted to clarify the clinical feasibility of cardiac MDCT in the evaluation of feline CAs.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Seoul National University . Written informed consent was obtained from the owners for the participation of their animals in this study.JK: setting the direction and design for the overall study and writing the main paper. D-HK: writing the thesis, analyzing and interpreting data, and reviewing the patients' conditions. KK: collecting and analyzing the patients' data and contributing to the direction of this study. DO: analyzing data and considering clinical aspects of the study. JY: coordinating the overall flow and direction of the study and writing and editing the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cardiac auscultation is a core clinical skill taught in medical school. Due to contact restrictions during the SARS-CoV-2 pandemic, interaction with patients was very limited. Therefore, a peer-to-peer virtual case-based auscultation course via video conference was established.rd-year medical students. A total of sixty medical students were randomly assigned to either the experimental or control group after informed consent was obtained. Due to no-shows, 55 students participated. Depending on allocation, students attended three ninety-minute courses in intervals of one week in a different order: a virtual case-based auscultation course held via video chat, literature self-study, and an on-site course using a high-fidelity auscultation simulator (SAM II). The study's primary endpoint was the performance of the two groups at the simulator after participating in the virtual auscultation course or literature self-study. To evaluate their auscultation skills, students participated in five assessments using the same six pathologies: stenosis and regurgitation of the aortic and mitral valve, ventricular septal defect, and patent ductus arteriosus. Moreover, participants rated their satisfaction with each course and provided a self-assessment of competence.A randomized controlled cross-over study was conducted to evaluate whether participation in a virtual auscultation course could improve heart auscultation skills in 3Compared to literature self-study, participation in the virtual auscultation course led to a significantly improved description of heart murmurs at the auscultation simulator with regard to the presence in systole and diastole, low- and high-pitched sounds, and volume dynamics. There was no significant difference between the groups in diagnostic accuracy and identification of the point of maximal intensity. After the virtual course, students showed higher satisfaction rates and a higher increase in self-assessed competence compared to participants who engaged in literature self-study.For the first time, this study demonstrates that a case-based virtual auscultation course can improve aspects of cardiac auscultation skills on a simulator. This may facilitate the further acquisition of an essential clinical skill, even when contact restrictions will be lifted. Cardiac auscultation using a stethoscope allows immediate identification of important cardiac pathologies. It is easily accessible and part of most physical examinations enabling skilled examiners to make an accurate diagnosis  or to inAuscultation is an essential examination technique. However, it requires the development of complex auditory skills. In medical education, these skills are taught through lectures, examination courses, and bedside teaching. Bedside teaching allows medical students to learn in a realistic environment, place heard heart sounds in context with other physical findings and recognize typical patterns of sound radiation across the thorax. However, infrequent exposure to rare findings as well as a high ratio of learners to patients pose challenges . MoreoveIn the past, many studies have shown poor auscultation skills of medical students , residenAddressing a generation of digital natives, the use of simulation technology and e-learning for skills training and assessment in medical education has progressively increased over the last two decades: Multiple simulation methods to train cardiac auscultation, summarized in a review of Ward and Wattier  and a meDuring the ongoing contact restrictions due to the SARS-CoV-2 pandemic, courses at the on-site skills lab with an auscultation manikin were suspended, and interaction with real patients in the context of bedside teaching was compromised . Similar1), higher diagnostic accuracy (H2), and more reliable identification of the point of maximal intensity (H3) of heart murmurs when compared to literature self-study, immediately after the course  and at follow-up at the simulator . The participant’s performance at the simulator was the study’s primary endpoint .According to results from previous studies , 20], , , 22], , , the fol4) ..4) [31].5) and a higher increase in self-assessed competence (H6) when compared to literature self-study.Following previous evaluation results , it was A prospective, randomized, controlled cross-over study was carried out at the Medical Faculty of the Heinrich-Heine-University of Duesseldorf, Germany. The study was designed to compare cardiac auscultation skills after participating in a one-time ninety-minute, virtual case-based auscultation course with participation in time-equivalent literature self-study.https://www.microsoft.com/de-de]), and an on-site course using a student auscultation manikin (SAM). Courses took place one week apart in groups of 5 (SAM) to 10 participants (VAC & SST).As shown in figure 1 Auscultation performance was assessed on five occasions (T1-T5) using the same six pathologies found in adult and pediatric patients: stenosis and regurgitation of the aortic and mitral valve, ventricular septal defect, and patent ductus arteriosus. The primary endpoint of the study was the performance at an auscultation simulator seven days after participating in one of two different interventions. Group A (exposed) participated in a virtual auscultation course (VAC), Group B (Control group) in literature self-study. Assessments T1, T2, T4, T5 were conducted before and after the VAC and SST to monitor any direct effect on the auscultation performance.Participant's satisfaction and self-rated competence were assessed using evaluation forms completed after every course (EV1-EV3).Approval for the study was obtained from the Ethics Committee of the Medical Faculty, Heinrich Heine University, Germany, (Nr. 2021-1298). The study was conducted in accordance with the Declaration of Helsinki.Sixty third-year medical students were recruited on a voluntary basis and provided informed consent to participate. Successful attendance was rewarded with 10€. The randomization process is shown in figure 2 Students participated in three courses  of equal length (90 min) in two different orders, depending on randomization. To avoid bias, all courses were held by the same tutor.The interactive online seminar was designed to improve listening technique, description, and interpretation of auscultation findings in an off-site context . This cahttps://clinisurf.elearning.aum.iml.unibe.ch/] developed at the University of Bern, Switzerland, were used.With kind permission, synthesized heart sounds, provided by the online learning program Clinisurf . To avoid confounding, the order of pathologies was identical following the study's cross-over design: Before VAC, SST and SAM and after VAC and SST, the same order was used for both groups.The order of the cardiac pathologies was randomized using the Research Randomizer , ,  to high-Considering these limitations, video conference-based auscultation courses may be a valuable addition to the already existing repertoire of auscultation training methods. Participation in training offered face-to-face or by video conferences can lead to similar levels of knowledge and confidence in healthcare professionals . This isConsequently, further research is needed to examine skill transfer from a virtual auscultation course to patients in a bedside scenario, possibly through the comparison of a virtual course with an auscultation simulator. The integration of bedside recorded heart sounds at multiple positions across the patient's thorax could improve the identification of typical patterns of sound radiation.Once established, virtual auscultation courses are easily accessible and transferable to other faculties, upscaling the number of courses does not require additional equipment and they are not affected by contact restrictions. The presented course design employed peer teaching and may therefore be more resource-efficient than formats relying on post-graduate teaching staff. Moreover, they allow for training auscultation even in remote areas with limited access to alternative training possibilities.Due to contact restrictions, the transfer of auscultation skills to patients in clinical practice has not been investigated in this study. However, good performance on an auscultation simulator correlated with good performance in real patients as shown by Fraser et al.  with regLong-time retention of auscultation skills was not assessed in this study. However, former studies have shown good long-term retention up to three years after training on an auscultation simulator , 21]. T. T21]. TMoreover, even though also the VAC was supervised, the participant’s screens could not be monitored for data protection reasons. Thus, it cannot be ensured that participants worked with the literature provided or followed the virtual course the entire time.The present study was sufficiently powered for valid conclusions, but more subtle effects of the VAC on auscultation performance with a moderate or small effect size may not have been detected.This study demonstrates for the first time that training medical students with a virtual auscultation course via video conference may significantly improve their heart auscultation skills after one week on a simulator when compared to literature self-study. The digital teaching format was rated as good by the participants and lead to a self-assessed increase in competence and satisfaction. Thus, even after contact restrictions are lifted, an interactive online course may provide additional benefit and support acquiring fundamental skills in heart auscultation.Bastian Malzkorn and Carsten Döing shared senior authorship.The authors declare that they have no competing interests.


Introduction: This systematic review with network meta-analysis aimed at comparing the medium-term results of open surgery (OS), fenestrated endovascular repair (FEVAR), and chimney endovascular repair (ChEVAR) in patients with juxta/pararenal abdominal aortic aneurysms (JAAAs/PAAAs). Materials and methods: MEDLINE, SCOPUS, and Web of Science were searched from inception date to 1st July 2022. Any studies comparing the results of two or three treatment strategies  on medium-term outcomes in patients with JAAAs/PAAAs were included. Primary outcomes were all-cause mortality, aortic-related reintervention, and aortic-related mortality, while secondary outcomes were visceral stent/bypass occlusion/occlusion, major adverse cardiovascular events (MACEs), new onset renal replacement therapy (RRT), total endoleaks, and type I/III endoleak. Results: FEVAR  was associated with higher medium-term all-cause mortality than OS. Sensitivity analysis including only studies that analysed JAAA showed that FEVAR  persisted to be associated with higher medium-term mortality than OS. Both FEVAR  and ChEVAR  were associated with a higher aortic-related reintervention rate than OS. No difference between different treatment options was found in terms of aortic-related mortality. FEVAR  and ChEVAR  were associated with a higher rate of medium-term visceral branch occlusion/stenosis compared to OS; however, there was no difference found between FEVAR and ChEVAR. Conclusions: An advantage of OS compared to FEVAR and ChEVAR after mid-term follow-up aortic-related intervention and vessel branch/bypass stenosis/occlusion was found. This suggests that younger, low-surgical-risk patients might benefit from open surgery of JAAA/PAAA as a first approach. The complexity of abdominal aortic aneurysm (AAA) repair depends mainly on anatomical detail relating to the segment of non-dilated aorta between renal arteries and the aneurysm, referred to as the aneurysm ‘neck’. Some 40–60% of aneurysms fall within the category of infrarenal AAA with adequate neck characteristics ,2, and tJuxta/pararenal abdominal aortic aneurysms (JAAAs/PAAAs) are a frequent variation of complex abdominal aortic aneurysms (AAAs). JAAA is defined by European Society for Vascular Surgery guidelines and Society for Vascular Surgery reporting standards as “AAA which extends up to renal arteries but does not involve them”, while PAAA is defined as “AAA where at least one or both renal arteries derive from AAA itself, but does not involve the superior mesenteric artery” ,5,6.Recent systematic reviews and meta-analyses have demonstrated the short-term benefit of fenestrated and chimney endovascular aneurysm repair (FEVAR/ChEVAR) in comparison with open surgery (OS) ; howeverThis systematic review and network meta-analysis aimed at comparing the medium-term results of OS, FEVAR, and ChEVAR for patients with juxta/pararenal abdominal aortic aneurysms.We performed a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and assessing the methodological quality of systematic reviews (AMSTAR) guidelines ,12. MEDLwww.rayyan.ai, accessed on 12 August 2022) was used for abstract screening.We included studies comparing the results of two or three treatment strategies  on medium-term clinical outcomes for patients with JAAAs/PAAAs. Medium-term was defined as a follow-up period of at least 6 months, ranging up to 60 months. Studies with standard infrarenal AAAs, AAAs with long but hostile neck characteristics  where standard infrarenal EVAR outside instructions for use has been performed, infected AAAs, ruptured AAA patients, thoracoabdominal aortic aneurysms (ThAAAs), suprarenal AAAs involving visceral segment at the level of the superior mesenteric artery and celiac trunk, connective-tissue-related aneurysms, studies containing less than 10 patients per treatment arm (to ensure enough experience in the treatment of this complex AAA pathology), performed before 2010 (to reduce the number of different stent graft generations in the comparison), and those having less than 6 months of follow-up were excluded from the analysis. Systematic reviews with or without meta-analysis, traditional reviews, comments, editorials and letters, and case reports were excluded, as well as any animal studies. A hand search of systematic reviews was also performed. Non-English articles were excluded unless they had an English abstract with extractable data. Two reviewers (P.Z. and P.T.) independently screened titles and abstracts as well as full texts of potentially eligible studies. Disagreements were resolved by discussion or consulting the third and fourth authors (A.J. and L.D.). The Rayyan systematic review web application  that were defined as a composite endpoint of cardiac death, myocardial infarction, coronary artery revascularisation, stroke, and hospitalisation because of heart failure; new onset renal replacement therapy (RRT); total endoleaks; and type I/III endoleak (including persistent gutter type Ia endoleak in ChEVAR group).No RCTs that fulfilled the inclusion criteria were found after the screening of the manuscripts. The Risk of Bias in Non-randomized Studies—of Interventions (ROBINS-I) tool was used to assess the quality of included observational studies . The GraA network meta-analysis (NMA) within a Bayesian framework was performed using WinBUGS14 software, with codes adapted from Dias et al. . The parA total of 1723 publications were identified, and after abstract screening, 63 were deemed relevant and read in full text. The network meta-analysis included 16 studies ,30,31,32The characteristics of the studies included in the network meta-analysis are presented in The overall quality of studies, as assessed by the ROBINS-I tool, was deemed “low”, with 10 studies (62%) having being deemed as having either serious or critical risk of bias in one or more domains ,26,27,31Values of the deviance information criteria (DIC) were similar in both models for all primary outcomes. However, lower values of residual deviance (Dres) were observed for the random-effects (RE) model compared with the fixed-effect model (FE) in all analyses. Seeing how both the values of Dres and the fact that studies were observational and heterogeneous were an indication for using the RE model, it was chosen in order for the estimate to be more conservative .Fourteen studies reported on all-cause medium-term mortality. This NMA included 4229 patients, and 417 deaths were reported (9.8%) a. The unEleven studies reported on aortic-related reintervention as an outcome. A total of 1497 patients were included in this NMA, with 146 patients who underwent aortic-related reintervention (11.2%) b. The unTen studies reported on aortic-related mortality. A total of 1150 patients were included in NMA, with 12 patients contributing to aortic-related mortality (1.1%) c. The unResults from NMA showed that FEVAR  and ChEVAR  were associated with a higher rate of medium-term visceral branch occlusion/stenosis compared to OS; however, there was no difference between FEVAR and ChEVAR in terms of this complication. No difference was found between the three treatment options in terms of renal replacement therapy and MACEs. When comparing FEVAR and ChEVAR, no difference was found regarding the total number of endoleaks and more malignant ones such as type I/III .This NMA found only observational studies comparing medium-term outcomes of interventions for JAAA/PAAA, mostly of low quality. The results of this NMA showed that FEVAR had a higher mid-term mortality compared to OS. Both endovascular procedures had higher rates of aortic-related reintervention and side branch occlusion/stenosis compared to the OS group. When making a comparison between the endovascular techniques, no significant preferences for either FEVAR or ChEVAR were found for any of the outcomes.In this meta-analysis, several attempts were made to ameliorate the limitations of the existing literature. Firstly, it is difficult to interpret the results of the existing meta-analyses due to anatomical heterogeneity of the patients ,33. We fCurrent guidelines recommend that the choice of different techniques and options for the management of JAAA/PAAA in the elective setting should be considered based on patient status, anatomy, local routines, team expertise, and patient preference ,5. The fAccording to recommendation 97 from the latest ESVS guidelines , FEVAR iThis NMA showed one interesting finding that FEVAR patients had worse medium-term all-cause mortality compared to the OS group. A similar tendency was found for ChEVAR patients compared to OS, but this did not reach the level of statistical significance. Due to the non-randomised nature of all studies included in this NMA, it is possible that this reflects a confounding from the variations in baseline clinical characteristics between the groups, but it is an important finding and an indication for future RCTs regardless. It is also possible that a confounding due to indication is present, i.e., that surgeons tend to choose endovascular solutions for less fit patients, and therefore, these solutions have worse medium-term outcomes. A general observation from A significantly higher rate of mid-term reinterventions in both the FEVAR and ChEVAR group compared to OS was demonstrated in this NMA. A recent NMA  showed tThe branch vessel occlusion/stenosis rate was lower in OSR. However, the interpretation of results should be taken with caution since most of the patients had juxtarenal AAA, and only 11.5% of all patients undergoing OSR had renal artery bypass/reattachment, which makes comparison to FEVAR/ChEVAR difficult. Surprisingly, there was no difference between FEVAR and ChEVAR in terms of incidence of new onset endoleaks and branch vessel patency. Most of the trials used company-manufactured stent grafts from Zenith Cook  for FEVAR and from Endurant  for ChEVAR. In the meta-analysis of Katsargyris et al. , no diffOne important factor that should not be neglected is the impact of centre volume on patient outcomes. As reported in a previous registry, centres with high (>14) volume of open JAAA repair demonstrated significant adjusted lower perioperative mortality (3.9%) compared to centres with low volume of open repair . PossiblThere are several concerns in this NMA. Our study included only observational studies and registries with significant differences in terms of baseline clinical characteristics. No RCTs have been performed comparing JAAA/PAAA repair. The GRADE rating for evidence was “low” for the majority of pairwise comparisons, reflecting the inherited bias. Data veracity is the Achilles’ heel of all retrospective analyses. Such nuances were most apparent in big registries . AnotherThe results of this NMA found an advantage for OS regarding aortic-related intervention and vessel branch/bypass stenosis/occlusion compared to FEVAR and ChEVAR after medium-term follow-up. This suggests that younger, low-surgical-risk patients might benefit from open surgery of JAAA/PAAA; however, this insight should be interpreted with caution due to the low quality of the included studies in the analysis and the possibility of confounding by indication, bearing in mind the observational design of the included studies. Further larger studies including experienced and high-volume AAA centres in patients with similar baseline patient characteristics are needed to adequately determine medium and long-term results of all three used treatment options.


Evidence regarding the learning curve of robot-assisted total mesorectal excision is scarce and of low quality. Case-mix is mostly not taken into account, and learning curves are based on operative time, while preferably clinical outcomes and literature-based limits should be used. Therefore, this study aims to assess the learning curve of robot-assisted total mesorectal excision.A retrospective study was performed in four Dutch centers. The primary aim was to assess the safety of the individual and institutional learning curves using a RA-CUSUM analysis based on intraoperative complications, major postoperative complications, and compound pathological outcome . The learning curve for efficiency was assessed using a LC-CUSUM analysis for operative time. Outcomes of patients before and after the learning curve were compared.In this study, seven participating surgeons performed robot-assisted total mesorectal excisions in 531 patients. Learning curves for intraoperative complications, postoperative complications, and compound pathological outcome did not exceed predefined literature-based limits. The LC-CUSUM for operative time showed lengths of the learning curve ranging from 12 to 35 cases. Intraoperative, postoperative, and pathological outcomes did not differ between patients operated during and after the learning curve.The learning curve of robot-assisted total mesorectal excision based on intraoperative complications, postoperative complications, and compound pathological outcome did not exceed predefined limits and is therefore suggested to be safe. Using operative time as a surrogate for efficiency, the learning curve is estimated to be between 12 and 35 procedures.The online version contains supplementary material available at 10.1007/s00384-022-04303-7. Surgical resection is the cornerstone of rectal cancer treatment. This is performed according to the total mesorectal excision (TME) principle, using minimally invasive approaches such as laparoscopic (L-TME), robot-assisted (R-TME) or transanal (TaTME) . MultiplHowever, outcomes of minimally invasive surgery are suggested to be influenced by the learning curve . CurrentDespite the increased number of papers published on this topic, studies are mostly of poor quality. Series are small and use different definitions for rectal carcinoma, or even include rectal resections for benign diseases as well. Additionally, different outcomes and statistical methods are used to assess the learning curve –22. MostThis is a retrospective multicenter cohort to assess the learning curve of R-TME in four large Dutch teaching hospitals. Learning curves will be assessed for individual surgeons and institutions. A protocol regarding the data-analysis was composed prior to initiation of the study. The manuscript was written according to the STROBE guidelines .This study involves four robot-assisted centres using the da Vinci system . None of the surgeons had prior experience with robot-assisted surgery, and start of the R-TME was preceded by electronic training, animal training, and proctoring of the first 5 procedures led by Intuitive. All surgeons had profound experience with more than 200 L-TMEs and more than 100 open TME procedures performed per surgeon. Centers started with the technique between 2011 and 2016. In the center, the A cases were operated using the DaVinci Xi, performed by one dedicated surgeon. Center B and C used the DaVinci Si, performed by two dedicated surgeons and a dedicated team of OR nurses per center. Center D used the DaVinci Xi, performed by two dedicated surgeons.All consecutive patients that underwent R-TME since its introduction in the specific center were included if they met the following criteria: (1) in need of TME, (2) diagnosed with rectal cancer according to the Sigmoid take-off definition , (3) werOur primary outcome was to assess the learning curve of the individual surgeons using clinical outcomes. The used clinical outcomes were as follows: intraoperative complications, postoperative major morbidity, and compound pathological outcome. Intraoperative complications were defined as injury to the bladder, ureter, urethra, or vagina, bleeding needing transfusion, serosal injury of the intestine, or any other complication needing intraoperative intervention. Postoperative major morbidity was defined as a postoperative complication within 30 days classified as Clavien–Dindo grade three or higher . The comOur secondary outcome was estimating the learning curve regarding efficiency, and comparison of outcomes between patients who underwent surgery during the learning curve and after the learning curve had been achieved. For efficiency, operating time was used, which was defined as time from incision until closure. Learning curves were assessed for both individual surgeons as well as institutions.Data was captured using the prospective DCRA database of the particular hospital, and missing or unregistered data was added from the local electronic medical record (EMR) system. Data was pseudonymized and registered in the GCP-proof data management system Castor . This study was approved by the regional medical ethics review committee , and the local hospitals’ medical ethics review committees.Patient characteristics that were captured from either the EMR or from the DCRA database included the following: age, sex, body mass index (BMI), ASA (American society of Anesthesiologist) classification, history of abdominal surgery, and history of pelvic surgery. Tumor characteristics included distance of the tumour from the anorectal junction (ARJ) on MRI in centimetres, a low rectal tumour classified according to the LOREC criteria , clinicaT-test or the Wilcoxon rank sum test for continuous variables, depending on the distribution. For categorical and binary variables, the X2 test was used. The learning curve was assessed using the risk-adjusted cumulative sum analysis (RA-CUSUM) for intraoperative complications, postoperative major morbidity, and the compound pathological outcome. For operating time, a learning curve cumulative sum analysis (LC-CUSUM) was used to assess the learning curve. A ‘normal’ (RA-)CUSUM assumes an ‘in-control’ state, and signals when the surgeon is out of control, whereas the LC-CUSUM assumes an ‘out-of-control’ state, and signals when the surgeon is in control. In addition, by using the risk-adjusted version, a poor outcome in a high-risk patient will be penalized differently compared to a poor outcome in a low-risk patient ), while major morbidity, intraoperative morbidity, and compound pathological outcome were comparable -CUSUM analyses for clinical outcomes with literature-based limits, involve studies evaluating TaTME surgery , 9, 12. ications , 9, 12. As clinical outcomes stayed within predefined, literature-based limits in this study, length of the learning curve could not be based on clinical outcomes. Therefore, we used operative time, as a secondary outcome, to assess length of the learning curve. The LC-CUSUM analysis showed length of the learning curve to range between 12 and 35 cases for individual surgeons. Previous studies assessing the learning curve based on operative time, using a limit are scarce. A R-TME study suggested a length of 19 cases, while a TaTME study suggested a length of 39 cases , 42. OutWhen comparing patients operated during the learning curve with patients operated after the learning curve, we saw a significant decrease in operative time. Several studies that based the learning curve on operative time, showed a significant decrease in operative time , 11, 38.To our knowledge this is the first study assessing the individual learning curves of R-TME surgeons by means of RA-CUSUM analyses of clinical variables, using literature-based limits. Although this study suggests that the learning curve of R-TME, for surgeons experienced in open or L-TME, is not associated with additional morbidity, and length of the learning curve for operating time is between 12 and 35 procedures, certain limitations should be taken into account. First, it should be appreciated that the learning ability and experience of the surgeons varies widely between surgeons, while statistical analyses cannot control for these variables. Therefore, these analyses reflect a theoretical simplification of a complex process. Second, this retrospective cohort analysis comes with reporting bias and confounding. We tried to control for confounding factors by using RA-CUSUM analyses for assessing the learning curve and a multivariable regression analysis when comparing outcomes during and after the learning curve. However, residual confounding might be present. Third, although most of the series were of considerable length, two surgeons only performed 30 and 27 procedures while operating times were missing in more than 50% of these cases. Therefore, we excluded these two surgeons regarding the LC-CUSUM analysis. Fourth, although we used literature-based limits for the RA-CUSUM and LC-CUSUM, especially the limit for operating time might be debatable. Not only case-mix influences this, as this is a variable reflecting efficiency which is affected by more than only the surgeon performing the procedure. In addition, cultural, logistic, and financial factors may differ between centers and could influence these outcomes as an effect. Fifth, a significantly higher rate of patients operated before the learning curve were treated with neo-adjuvant therapy, due to change of the Dutch Guideline in 2014 . This miConcluding, this is the first study assessing the learning curve of R-TME by means of a RA-CUSUM analysis with literature-based limits, using clinical variables. This study suggests that the learning curve of R-TME is safe in terms of intraoperative complications, major postoperative complications and pathological outcomes. The learning curve based on efficiency might range between 12 and 35 procedures for R-TME.Supplementary file1 (DOCX 31 KB)Supplementary file2 (DOCX 32 KB)Supplementary file3 (DOCX 31 KB)Supplementary file4 (DOCX 20 KB)Below is the link to the electronic supplementary material.


Few studies have examined the association of geography and quality of life (QOL) among breast cancer patients, particularly differences between Appalachian and non-Appalachian Kentucky women, which is important given the cancer and socioeconomic disparities present in Appalachia.The purpose of this study was to determine whether women with breast cancer residing in Appalachian Kentucky experience poorer health outcomes in regards to depression, stress, QOL, and spiritual wellbeing, relative to those living in non-Appalachian Kentucky after adjusting for demographic, socioeconomic, and health-related factors.Women, aged 18–79, recruited from the Kentucky Cancer Registry between 2009 and 2013 with an incident, primary breast cancer diagnosis completed a telephone interview within 12 months of diagnosis. In this cross-sectional study, sociodemographic characteristics and mental and physical health status were assessed, including number of comorbid conditions, symptoms of depression and stress, and QOL.Among 1245 women with breast cancer, 334 lived in Appalachia and 911 in non-Appalachian counties of Kentucky. Appalachian breast cancer patients differed from non-Appalachian patients on race, education, income, health insurance status, rurality, smoking, and stage at diagnosis. In unadjusted analysis, Appalachian residence was associated with having significantly more comorbid conditions, more symptoms of stress in the past month, and lower Functional Assessment of Cancer Therapy-Breast scores compared to non-Appalachian residence.However, adjustment for sociodemographic and health-related differences by region appear to explain geographic differences in these poorer QOL indicators for women living in Appalachian Kentucky relative to non-Appalachian Kentucky. Policy-, provider-, and individual-level implications are discussed. Once women were reached via telephone, the interviewer obtained verbal consent before beginning the interview. The average interview duration was 30 minutes; women were offered a $10 incentive.Varying demographic, socioeconomic, and health-related items were included in the questionnaire to create a profile of female breast cancer patients by region in Kentucky. Stage at diagnosis , age at diagnosis, health insurance status, and county of residence were available from KCR. Each county’s corresponding 2013 Rural–Urban Continuum Code was used to create a rural–urban classification. County of residence was also used to create the dichotomous independent variable of Appalachian or non-Appalachian.Dependent variables included: (1) comorbid physical conditions at diagnosis; (2) symptoms of depression and stress; and (3) QOL and spiritual wellbeing. Women were asked whether a doctor had ever told them they had additional health conditions . Response options for each condition were yes or no. Physical conditions were summed to create an ordinal variable indicating the number of conditions the woman has experienced.Symptoms of stress were determined using three of the four-item Perceived Stress Scale (PSS). Participants were asked to use this scale to recall perception of stress during the two to three months after initial diagnosis as well as stress the month prior to the interview. Replies were measured on a five-point Likert scale (0=Never…4=Very Often). The Cronbach alpha for the altered PSS measure was 0.63 and 0.60 for the recall periods, respectively.10Cancer-related QOL was measured with a 27-question Functional Assessment of Cancer Therapy-Breast Cancer questionnaire .10Sociodemographic and health attributes of breast cancer cases living in Appalachian and non-Appalachian regions were compared to determine covariates for subsequent analyses addressing the associations between geographic region and QOL cancer outcomes using either two-sample t-test for the two continuous measures (age at diagnosis and number of children) or chi-square tests for the remaining characteristics . In an eANOVA analyses were used to compare outcomes for Appalachian (exposed) versus non-Appalachian (non-exposed) residence; adjustments for covariates were made using ANCOVA. These analyses were performed separately for the dependent variables of number of covariates, total FACT-B score, and FACIT-Sp. A similar analysis was performed using MANOVA without adjustments and MANCOVA with adjustments for dependent variables for stress and depression as well as for the domains of the FACT-B score because these outcomes were correlated. For models using each outcome variable, the t-statistic, df, and p-value for the effect of Appalachian residence is provided. Analyses were completed in 2016 using in SAS® Version 9.3 (Cary NC); p-values ≤ 0.05 were considered statistically significant.Of the 4628 women diagnosed with breast cancer and included in the KCR between November 2009 and December 2013, we could not survey 1414 women (30.6%) because 42 had died, physicians requested that 24 cases not be contacted, and 1348 could not contacted by phone or mail. Another 1969 women (42.5%) refused participation. Response rates (n=1245) were 26.9% of all 4628 women diagnosed with breast cancer, or 38% of 3280 we contacted for active consent. Response rates were higher among Appalachian Kentucky women (29.9%) than non-Appalachia women (25.9%) .Women with breast cancer who lived in Appalachian Kentucky differed from those living in non-Appalachia on race, education, income, health insurance status, rurality, smoking, and stage at diagnosis . Among Adf 3,1237p=0.08) and the Wilks’ for MANOVA for the four FACT-B subscales was significant . In the unadjusted model (model a), Appalachian residence was associated with more comorbid physical conditions, more symptoms of stress in the past month, and poorer cancer-related QOL as measured with the total FACT-B score and all but the social domain for FACT-B subscales. No regional differences in symptoms of depression or stress at diagnosis were observed; similarly, FACIT-Sp scores did not differ by region. These patterns generally held when adjusting for age at diagnosis, rurality, race, and stage (model b), yet were not significant when additionally adjusting for income and private insurance (modelc). The addition of smoking and education to the final model (model d) suggests that these two covariates did not explain patterns beyond adding income and insurance in the prior models (model b and c).Appalachian women reported more physical comorbidities, higher stress levels at diagnosis and within the past month, and a lower FACT-B total score  indicating decreased QOL as compared to non-Appalachian patients . FurtherTo our knowledge, this is one of the first studies to specifically explore cancer-related QOL differences between women with breast cancer residing in Appalachian versus non-Appalachian Kentucky. We found that Appalachian women were more likely to live in extremely rural communities, be of lower socioeconomic status (SES), and experience poor health outcomes such as higher rates of smoking, Stage 4 disease, physical comorbidities, and stress compared to their non-Appalachian counterparts. In reviewing the unadjusted mean scores, Appalachian women also had lower FACT-B total scores (2.55-point difference). However, after adjustment for sociodemographic and cancer attributes, women living in Appalachian Kentucky did not have poorer cancer-related QOL compared to women residing in non-Appalachia. Adjustment for age at diagnosis, rurality, race, stage, income, and insurance status appear to mediate or explain regional differences in cancer-related QOL noted in the unadjusted comparisons. Specifically, income and private insurance are likely the important mediators explaining Appalachian regional differences in cancer-related QOL because their addition to models resulted in no observed regional differences in the noted outcomes. These findings support Schootman et al. who found geographic differences in rates of depression and social support were not significant once SES, access to medical care, or other chronic conditions were included in the analysis.11Overall, the findings reiterate the powerful influence of SES on breast cancer outcomes, including QOLAlthough this study is a unique contribution to the breast cancer QOL literature, particularly its focus on Appalachia, there are noted limitations in the cross-sectional methodology. A primary limitation is collecting several of the sociodemographic variables and defining QOL based on women’s self-report, which may be biased; yet women are the ultimate authority on their own QOL and mental health. Those completing interviews (38% of women we were able to contact) may differ from those who did not participate on attributes we could and could not measure. For example, KCR did not provide specific data on stage or age for those women who did not complete the survey. We were able to document that Appalachian women were more likely to agree to be interviewed than those living in non-Appalachia; however, this modest difference is unlikely to bias the consistently null findings observed here. Literature comparisons were generated from U.S. rural versus urban cancer QOL studies, which may not translate directly to Appalachian and non-Appalachian areas of Kentucky. Study limitations are countered with strengths, including use of the same interview protocol for all participants and use of outcome measures with strong psychometric properties, thereby limiting measurement bias. Sampling from KCR improved study power and sample representativeness. Moreover, the study provides a foundation for future research examining psychological and other predictors of breast cancer-related QOL outcomes in Kentucky as well as the entire 13- state Appalachian region, including assessments of rural and urban counties, Appalachian subregions, and non-Appalachian areas.What is already known about this topic?Rural-residing breast cancer patients have previously reported higher levels of stress and hopelessness, lower quality of life (QOL) and lower functional wellbeing, and increased symptom complaints.What is added by this report?Few studies have specifically examined differences in QOL between Appalachian and non-Appalachian Kentucky women diagnosed with breast cancer. Adjustment for sociodemographic and health-related outcomes by geographic region appear to explain differences in poorer QOL indicators for women in Appalachian Kentucky relative to non-Appalachian Kentucky.What are the implications for public health practice, policy, and research?Socioeconomic status (SES) is a powerful influence on breast cancer outcomes, including QOL. Additional research is needed to understand the complex interplay between SES, geographic residence, mental health status, and cancer.


The ongoing COVID-19 pandemic caused by SARS-CoV-2 has changed everyday life worldwide. To reduce disease transmission, governments introduced various policies such as social distancing, stay-at-home orders, and travel restrictions. The goal of this study was to investigate the characteristics of burn patients admitted to the burn intensive care unit before and during the COVID-19 pandemic.A retrospective descriptive analysis of the hospital's burn registry was performed from 1 March 2019 until 1 January 2022.A total of 326 patients were included in this study. Eighty-eight patients presented before and 238 patients during the COVID-19 pandemic. The majority of burns occurred during private incidents , and burns were most frequently caused by flames . Work-related injuries occurred less frequently . Constant results were obtained regarding severity and total body surface area affected (1–80%).This study highlights high numbers of burn patients admitted to the burn intensive care unit before and during the COVID-19 pandemic. Consequently, burn intensive care units must retain their special position within the national health system and should not be included in resource relocation during the prioritisation of intensive care resources. Multicentre studies should focus on the national impact of COVID-19 on the treatment of burn patients. The ongoing COVID-19 pandemic caused by SARS-CoV-2 has significantly changed everyday life worldwide since it was first reported in January 2020.A retrospective descriptive analysis of the hospital's burn registry was performed from the 1 March 2019 until the 1 January 2022. The register is maintained by the Department of Plastic, Aesthetic, Hand and Reconstructive Surgery of Hannover Medical School.Data of all patients who were admitted and treated in the burn intensive care unit of the department were collected anonymously using an online data collection portal. The required data were entered by the attending physician. An informed consent form was signed by the patients or their legal representative. As this was a retrospective analysis of an anonymous database, approval by the local ethics committee was not required.Current burn guidelines require a referral to a specialised burn unit in case of partial-thickness burns > 10% total body surface area (TBSA); full-thickness burns; burn injury of the hand, face, or genitalia; electrical injury; chemical burns; inhalation injury; or burn injury of paediatric patients.Data were collected from the hospital's internal database anonymously, including age at presentation, gender, date of trauma, height, weight, mechanism of burn injury, comorbidities, TBSA affected, presence of inhalation trauma, Abbreviated Burn Severity Index (ABSI), primary or secondary referral, and traumatological injuries. The primary assessment followed by the diagnosis of the burn injury, including calculation of the affected surface and burn depth, was performed by the attending physician and by a board-certified burn surgeon upon arrival. To detect further deepening of the burn wounds, daily re-evaluation was performed. Cases with unknown or missing data were excluded.Statistical analysis was conducted using GraphPad Prism , Microsoft Excel , and Numbers . Descriptive statistics are presented as numbers (percentage) and medians (interquartile range). The number of patients and their characteristics were compared using a two-way analysis of variance (ANOVA). Further, Tukey's and Šídák's multiple comparisons tests were applied. A p-value < 0.05 was considered statistically significant.A total of 326 patients were included in this study. Eighty-eight patients presented before the COVID-19 pandemic, whereas 238 patients suffered burn injuries during the pandemic Fig. 1The most common comorbidities of the admitted patients are summarised in This descriptive study investigated the detailed characteristics of burn patients admitted to a specialised centre in Northern Germany before and during the COVID-19 pandemic. Despite the efforts made by the government issuing policies, such as social distancing, city lockdowns, and stay-at-home orders, a continuously high number of burn patients admitted to the burn intensive care unit was observed.,Similar studies were published by other burn centres from economically developed countries such as the UK, the USA, Canada, and Japan.The major cause of private burn injuries was reported as flame burns, which is most likely explained by the prolonged stay of people in closed spaces in their homes. Similar results were reported in the literature.,Due to lockdown policies, work-related burn injuries were expected to decrease; however, our study did not identify statistically significant results. This might be explained by the fact that the lockdown policies mainly affected the retail, culture, and food sectors and spared the heavy industry where hygiene concepts or short-time working was introduced early in Germany. No relationship could be identified in previously published studies, which may be explained by the generally lower incidence of work-related burn injuries.The first wave of the COVID-19 pandemic put the global medical system to an especially hard test. Being confronted with a large number of critically ill patients, restructuring clinical pathways and services was necessary in order to maintain high standards of patient care. New consulting approaches via telemedicine follow-up proved to be a reliable tool for increasing patient-led self-care, reducing hospital admissions, and minimising clinical attendance of medical personnel.Our study highlighted continuously high numbers of burn patients admitted to the burn intensive care unit before and during the COVID-19 pandemic. Burn injuries represent a small yet complex patient collective that requires highly specialised intensive care staff and infrastructure as an intensive care unit. Consequently, the burn intensive care unit must maintain a special position within the national health system. Decision-makers must be discouraged from moving medical personnel from burn intensive care units to other specialised units since the incidence of burn patients was not affected for this patient cohort. As soon as the SARS-CoV-2 infection rate declines, resources should be reallocated to burn intensive care units in order to provide the highest standard of care for critically ill burn patients.This study has several limitations. It was retrospective study with a relatively small sample. This study also represents a single-centre analysis; however, it is the only specialised centre in the whole county, making the data still representative of a large geographic area. Our centre was confronted with a severe shortage of rehabilitation clinics, making secondary patients transfer the bottleneck for new patient admission; hence, the sample size is potentially biased and might be considered larger.The ongoing COVID-19 pandemic has dramatically changed everyday life around the world.Despite the efforts made by the government issuing policies, such as social distancing, city lockdowns, and stay-at-home orders, a continuously high number of burn patients admitted to the burn intensive care unit before and during the COVID-19 pandemic was observed.Flame burns caused the majority of burn injuries, most likely due to the prolonged stay of people in their homes. Further, work-related burn injuries remained constant as lockdown policies mainly affected retail, culture, and food sector while sparing the heavy industry. The expected rise in burn patients with concomitant Sars-CoV-2 infection did not set in, as only two burn patients were tested positive for COVID-19.The results of this study underline the special position of burn intensive care units within the national health system, being small, yet highly specialised departments. The COVID-19 pandemic has shifted the decision-makers focus away from the critical burn patients, making it necessary to highlight the importance of the highly specialised intensive care units.These units should not be included in prioritisation measures, and for providing the highest standard of care, resources should be relocated.There was no funding source for this study.Being a retrospective database analysis with anonymous patient data, no approval by the Ethics Committee was required according to the Institutional Review Board.Written informed consent was obtained from all subjects involved in this study.Data were collected from the hospital's internal database anonymously for each patient. After informed consent is obtained, the attending physicians will enter the above-mentioned data into a database. It can then be extracted anonymously for research purposes.F. Bucher: Conceptualization, Funding acquisition, Formal analysis, Writing – original draft. K. Dastagir: Software, Formal analysis, Visualization, Data curation. D. Obed: Methodology, Writing – review & editing. T. Dieck: Supervision, Data curation, Validation, Writing – review & editing. P.M. Vogt: Conceptualization, Supervision, Writing – review & editing, Project administration.The author and co-authors declare that they have no competing financial interests or personal relationships that could have influenced the results reported in this manuscript.