MYB28 transcription factor from the wild species Brassica villosa. Through a novel k-mer approach, we characterised the extent of the introgression of unique B. villosa genome sequences into high glucosinolate broccoli genotypes. RNAseq analyses indicated that the introgression of the B. villosa MYB28 C2 allele resulted in the enhanced expression of the MYB28 transcription factor, and modified expression of genes associated with sulphate absorption and reduction, and methionine and glucosinolate biosynthesis when compared to standard broccoli. A adenine-thymine (AT) short tandem repeat (STR) was identified within the 5′ untranslated region (UTR) B. villosa MYB28 allele that was absent from two divergent cultivated forms of Brassica oleracea, which may underpin the enhanced expression of B. villosa MYB28.Broccoli cultivars that have enhanced accumulation of methionine-derived glucosinolates have been developed through the introgression of a novel allele of the  Brassica oleracea, n = 9, species complex comprises several agronomic varieties and a series of partially sexually compatible wild perennial “species” that are found mainly on Mediterranean coastal cliff faces and associated habitats   . DespiteBrassica crops accumulate sulphur-containing glycosides commonly known as glucosinolates in vegetative and reproductive tissues. The glucosinolate molecule comprises a common sulphated aldoxime with a variable side chain derived from a small number of amino acids. Following tissue disruption, glucosinolates are hydrolysed to an array of bioactive compounds. Many of these hydrolytic products have been shown to mediate plant-herbivore and plant-pathogen interactions. The hydrolytic products of 2-hydroxy-3-butenyl glucosinolate that accumulates in the seeds of oilseed brassicas have goitrogenic activity when the protein-rich meal is fed to cattle, which led to the breeding and widespread adoption of low seed glucosinolate cultivars. Certain glucosinolates within horticultural brassicas, and in particular, 4-methylsulphinylbutyl glucosinolate (“glucoraphanin”) and 3-methylsulphinylpropyl glucosinolate (glucoiberin) derived from methionine and which accumulate in B. oleracea var. Italica (broccoli), have been associated with the putative health-promoting properties of cruciferous vegetables.Oilseed and horticultural B. oleracea have been used to explore the genetic basis of the chemical structure of the glucosinolate side chains. A small set of Mendelian genes interact to determine the length and chemical structure of the glucosinolate side chains. B. villosa, a wild form of B. oleracea found in Sicily, has been used as a source of a “high glucoraphanin” trait for broccoli breeding programmes for use in studies to explore the health-promoting activities of broccoli  have been previously described, and low-density KASpar markers were used to identify regions of genomic introgression of B. villosa into the broccoli genetic background  and the suppression of changes in oncogenic gene expression in prostate biopsy tissue. Men who consumed a broccoli soup manufactured from the standard broccoli cultivar Ironman (genotype MYB28 B/B) exhibited changes in gene expression in prostate tissue consistent with enhanced cancer risk, which was partially suppressed with Beneforte (MYB28 B/V) and entirely suppressed with a non-commercial broccoli genotype high-glucoraphanin (HG) Inbred, which was homozygous for the B. villosa allele (MYB28 V/V) (A 12-month dietary intervention study with men who had organ-confined prostate cancer demonstrated a dose-dependent association between the B28 V/V) .MYB28 V allele, it is conceivable that additional non-target introgressed genomic segments may have been of importance, particularly, in the HG Inbred genotype that completely suppressed changes in oncogenic gene expression. Moreover, the role of the additional two paralogues of MYB28, on chromosomes C7 and C9, in contributing to the high glucosinolate trait has not been determined. In the current study, we quantify through genomic sequencing and K-mer analyses the extent and distribution of the introgression of the B. villosa genome into HG Inbred and the agronomically superior high glucoraphanin F1 broccoli hybrid 1086, both of which are homozygous for the B. villosa MYB28 C2 allele. Furthermore, through RNAseq, we seek to investigate the consequences of the enhanced expression of MYB28 on gene expression associated with glucosinolate biosynthesis. Lastly, we speculated as to a possible regulatory element that results in enhanced expression of the B. villosa allele.Whilst it was assumed that the high glucosinolate trait and the suppression of changes in gene expression in prostate tissue were due to the introgression of the 1 hybrid broccoli that is homozygous for the MYB28 C2 broccoli allele (B/B). (2) 1199 (tradename Beneforte), an F1 hybrid that is heterozygous for the MYB28 C2 B. villosa/broccoli allele (V/B). (3) HG Inbred, an inbred genotype that is homozygous for the B. villosa MYB28 C2 allele (V/V). (4) 1086, an elite broccoli F1 hybrid that is homozygous for the MYB28 C2 B. villosa allele (V/V). Whilst both 1086 and HG Inbred have the same MYB28 C2 genotype (V/V) but the genetic background is different with 1086 exhibiting an elite broccoli agronomic phenotype in contrast to HG Inbred. These four genotypes were grown under normal agronomic conditions in an experimental field plot in Norwich in 2017. Leaves were sampled from three plants of each genotype for genome sequencing and gene expression analyses, and florets were sampled for metabolite analyses. All leaf material was harvested whilst the plants were in a vegetative stage prior to the emergence of inflorescences. Additional leaves were sampled from a single glasshouse-grown B. villosa plant for genomic and gene expression analyses.Four broccoli genotypes were used within the research project: (1) Ironman, a widely grown commercial F1 producing an average of 125 million total reads per sample with 35X average depth coverage  (Contigs from the CLC draft assemblies were anchored using gene models of the reference genome of on 97.1) . Coding on 97.1)  with defon 97.1) .B. villosa (donor) and Ironman (background) using jellyfish3 version 2.1.4. (B. villosa) per contig. For each contig that was able to be anchored to the TO1000 B. oleracea assembly (k = 31) was recorded. Subsequently, it was determined how many of those k-mers were present only in the raw data of the “donor” B. villosa and how many were present only in the raw data of the commercial broccoli background, Ironman. The resulting plot displays the ratio of k-mer alignment between these lines in which k-mer alignment with the B. villosa appears in black.K-mers were counted in raw-data of n 2.1.4. . The parassembly , this raB. villosa leaf of similar developmental stage. In Arabidopsis, leaves are the site of the biosynthesis of aliphatic glucosinolate that is subsequently transported to roots and reproductive tissues  were used to generate TruSeq non-directional RNA libraries at Earlham Institute (United Kingdom). Libraries were sequenced in one pool of seven (7-plex) and run on two lanes of the Illumina HiSeq2500 with a 125 bp paired-end read metric that generated an average of 160,170 million reads per sample. Sequencing was performed by the Earlham Institute. Individual plant sample RNA was used to generate 7 TruSeq non-directional RNA libraries. These libraries were sequenced through pooling in one pool of 7 (7-plex) and run on 2 lanes of the Illumina HiSeq2500 with a 125 bp paired-end read metric with an average of 160–170 million reads per sample. This data are available on NCBI under Project ID PRJNA623495.Transcriptome analysis was undertaken on vegetative leaves from field-grown individual plants of field-grown Ironman and HG Inbred in addition to a glasshouse-grown  tissues . Likewisemselves . RNA extp < 0.05) genes that was generated using the Ballgown software included in the “Tuxedo Suite” for RNA-seq data analysis (MYB28 V/V broccoli when compared to standard broccoli cultivar Ironman MYB28 B/B (p < 0.05). This data set was processed using TopGo analysis to gain a list of enriched gene ontologies in the dataset  (t-tests of Transcripts per Kilobase Million (TPM) in the RNA TPM in the RNA-seq analysis when comparing expression in the standard broccoli with the HG Inbred  . Differeanalysis , which p dataset . The genn 3.2.3) , in thisn 3.2.3) . A MultiG Inbred .APS3 (Bo5g021810), MAM C7 (Bo7g098000), and CYP79F1/F2 (Bo5g021810), were additionally quantified by quantitative reverse transcription PCR (RT-qPCR). MAM C7 has previously been referred to as MAM3  analyses were performed as described previously with minor modifications . GlucosiMYB28 V alleles had significantly greater total content of the aliphatic methionine-derived glucosinolates glucoraphanin and glucoiberin in their florets  and one accession of B. oleracea reference assembly of TO1000 (k = 31) was determined. We additionally obtained all k-mers from the raw data of Ironman and B. villosa , indicative of its greater divergence from TO1000 when compared to broccoli. 3286 genes were differentially expressed between Ironman and HG Inbred (uncorrected p < 0.05). Of the three MYB28 paralogues on C2, C7, and C9, only the MYB28 C2 allele was differentially expressed between Ironman and HG Inbred, with levels of expression in HG Inbred similar to that in B. villosa  was found in the sequence coding of the R2R3 DNA-binding domain  was situated within the 5′ untranslated region (5′UTR), and that the significant difference in sequence prevented the mapping of the broccoli and B. villosa sequence data to TO1000. It was also found that the AT STR was absent from the domesticated broccoli B. oleracea reference sequence DH1012, in a similar manner to Ironman .In addition to the importance of Brassica . There wB. villosa into the high glucoraphanin genotypes, although it appeared to have no phenotypic effects apart from enhancing aliphatic glucosinolates, the precision of the analyses was significantly compromised by the availability of broccoli genotypes. Ironman was used as a surrogate for the actual genetic background of the high glucoraphanin genotypes. Likewise, the B. villosa genotype used in the current analyses is not the precise same genotype used in the original cross with cultivated B. oleracea. This may account for the difference in the length of the AT STR near the ATG start codon, as discussed below  can be found below: National Center for Biotechnology Information (NCBI) BioProject database under accession number 1 hybrids and provided advice on broccoli breeding. MHT, LØ, and RM devised the research programme and provided oversight for all experimental work and analyses. MN, BS, and RM prepared the manuscript. All authors contributed to the article and approved the submitted version.MN undertook laboratory experimental studies, RNA-seq analysis, and field work. BS provided expertise in genome sequencing, K-mer analyses, and bioinformatics. PT-R and MHT provided expertise in RNA-seq analyses and gene ontology analyses. SS provided expertise and oversight for biochemical analyses. FB provided broccoli breeding genotypes and FThe broccoli with elevated glucoraphanin is the subject of patents filed by Plant Bioscience Limited (PBL), the technology transfer company of the John Innes Centre. RM and MHT are inventors named on these patents. FB was employed by Bayer. The remaining 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.


Reported evidence of coronary stent fracture (CSF) has increased in recent years. The purpose of this study was to determine reliable estimates of the overall incidence of CSF.The MEDLINE, Embase and Cochrane databases were searched until March 18, 2022. Pooled estimates were acquired using random effects models. Meta-regression and subgroup analysis were used to explore sources of heterogeneity, and publication bias was evaluated by visual assessment of funnel plots and Egger’s test. Overall, 46 articles were included in this study. Estimates of CSF incidence were 5.5% [95% confidence interval (CI): 3.7–7.7%] among 39,953 patients based on 36 studies, 4.8% (95% CI: 3.1–6.8%) among 39,945 lesions based on 29 studies and 4.9% (95% CI: 2.5–9.4%) among 19,252 stents based on 8 studies. There has been an obvious increase in the incidence of CSF over the past two decades, and it seems that the duration of stent placement after stent implantation has no impact on incidence estimation.The incidence of CSF was 5.5% among patients, 4.8% for lesions and 4.9% for stents and increased over the past 20 years. The duration of stent placement after stent implantation was found to have no impact on the incidence of CSF, but drug-eluting stent (DES) types and right coronary artery (RCA) lesions influenced the pooled incidence.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022311995], identifier [CRD42022311995].[ Coronary drug-eluting stents (DES) are widely used in the interventional treatment of coronary heart disease. The material, structure, technology, and thickness of the steel beam and the drug loading model of the stent have changed significantly . With thEspecially in recent years, the thinner steel beam thickness in the new generation of alloy stents and the wide use of intraluminal imaging technology have led to a significant increase in the incidence of CSF in clinical practice –5. In adTherefore, a systematic review and meta-analysis were performed to highlight the incidence of CSF and to summarize the current knowledge of related factors for CSF. We hypothesized that we could identify the incidence of CSF and the risk factors for CSF.1This systematic review and meta-analysis were performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-Analysis of Observational Studies in Epidemiology (MOOSE) guidelines. The complete details for this meta-analysis, including the electronic search strategy, objectives, criteria for study selection, eligibility, data collection, and assessment of study quality, are available in the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42022311995) on 25 March 2022.We searched the Cochrane Central Register of Controlled Trials , MEDLINE, and Embase databases from the inception dates to 18 March 2022 using keywords and Medical Subject Headings (MeSH) terms relevant to the exposure of interest to collect data for the systematic review and meta-analysis: “Strut Fracture,” “Strut Fractures,” “Strut Fractured,” “Strut Fracturing,” “Strut Crack,” “Strut Cracked,” “Strut Cracks,” “Strut Cracking,” “Stent Fracture,” “Stent Fractured,” “Stent Fractures,” “Stent Fracturing,” “Stent Crack,” “Stent Cracked,” “Stent Cracks,” “Stent Cracking,” “DES Fracture,” “DES Fractures,” “DES Fractured,” “DES Fracturing,” “DES Crack,” “DES Cracked,” “DES Cracks,” “DES Cracking,” “BMS Fracture,” “BMS Fractures,” “BMS Fractured,” “BMS Fracturing,” “BMS Crack,” “BMS Cracked,” “BMS Cracks,” and “BMS Cracking.” There were no language restrictions. Clinical randomized trials, controlled before-and-after studies, case control studies, and prospective and retrospective cohort studies were eligible for inclusion in the review. Cross-sectional studies, repeat publications of the same analysis or dataset, case reports, conference abstracts, opinion pieces, books or gray literature were excluded. To acquire qualitative/quantitative analyses, inclusion and exclusion criteria were summarized according to the PICOS  approach . Two resThe primary outcome of this meta-analysis was the incidence of CSF. CSF was considered and recorded in the research analysis according to reports of the use of plain fluoroscopy, computed tomography (CT), coronary angiography (CAG), intravascular ultrasound (IVUS) or optical coherence tomography (OCT) in the selected studies. The review was performed in accordance with instructions given in the Cochrane Handbook for Systematic Reviews of Intervention.Quality was assessed using the Cochrane Collaboration’s tool for assessing the risk of bias in randomized controlled trials (RCTs). For non-RCTs, the modified Newcastle–Ottawa quality assessment scale was used to assess the quality of the included cohort studies and case control studies. The total score for the modified Newcastle–Ottawa scale for cohort studies is a maximum of nine stars overall with a minimum of zero. A study was considered high quality if it achieved 7 out of 9 stars and moderate if it achieved 5 out of 9 stars . OverallP < 0.10. We expressed the percentage of heterogeneity attributable to variation rather than to chance as I2  to calculate the combined proportion value and 95% confidence interval by excluding each included study separately.Review Manager  and the R package for Windows (version 4.1.0) (meta package) were used to perform all the meta-analyses. Random effects meta-analyses were performed using the proportions of patients who experienced CSF as the outcome of interest. Because of the anticipated high degree of heterogeneity, predominantly among non-RCTs, an inverse variance (DerSimonian–Laird) random effects model was applied. Following the identification of each study to be included, precise event rates were noted from the reported results in all cases. After transforming the event parameters to a normal distribution, an appropriate estimation method was chosen to estimate the original rate . Pooled From the systematic review and meta-analysis search, 4,766 potentially eligible records were identified. Titles and abstracts of these records were screened for inclusion. The full texts of 73 records were read, and 46 studies were finally included in the data assessment , 11–50. The risk of bias of the included studies is summarized in the P = 0.5619, P = 0.0690, P = 0.0008)  –8.The pooled incidence rate for CSF from 36 studies with 39,953 subjects  was 5.5% (95% CI: 3.7–7.7%) using a random effects model because of a high level of heterogeneity (98%) . In compp < 0.01) (The CSF incidence rates for sirolimus-eluting stents (SESs), paclitaxel-eluting stents (PESs), everolimus-eluting stents (EESs), and biolimus-eluting stents (BESs) were 6% (95% CI: 3–8%), 0% (95% CI: 0–0%), 2% (95% CI: 0–5%), and 6% (95% CI: 4–7%), respectively. Tests for subgroup differences in DES type with random effects showed remarkable differences ( < 0.01) .The incidence rate of CSF at the patient level was not significantly different when the follow-up evaluation was executed with IVUS, OCT or CT  compared to when the follow-up evaluation was executed with plain fluoroscopy or CAG  .Overall, the pooled estimate for the subgroup including randomized studies differed appreciably from the combined result from observational studies. The CSF pooled incidence in randomized studies was 1% (95% CI: 0–2%); in observational studies was 6% (95% CI: 4–9%) .P = 0.8821, P < 0.0001, Generally, pulsation of the heart can result in metal fatigue and CSF. We evaluated the duration after stent implantation and the incidence of CSF with meta-regression. In the meta-regression analyses, patients’ overall CSF estimates were not modified by the duration of follow-up, which lasted for nearly 5 years . Second, the pooled CSF incidence rates of SES and EES were 6 and 2%, respectively. Third, the duration of the stent after stent implantation seemed to have no significant effect on the incidence of CSF. Fourthly, the incidence of CSF increased with time in recent decades. Finally, RCA lesions contributed significantly to CSF at the lesion level.To our knowledge, coronary DES fracture was first reported in 2004 , and thePrevious studies , 52, 54 IVUS and OCT have been used as more sensitive methods for the detection of stent fracture due to their high resolution , 58, 59.The quality control data of coronary stents has shown that stents can be used consistently for more than 10 years when they are expanded to the maximum diameter on the condition of simulative physiological heart beats , and no fatigue or fracture occurs. Combined with our analysis results of the association between the duration of the stent after stent implantation, it may be unnecessary for patients with stent implantation to undergo longer repeated evaluations for the occurrence of CSF. However, the best time range to detect CSF remains undefined.In general, there is greater curvature and range of motion in the right coronary artery (RCA) than in the left coronary artery, especially in the proximal-to-middle segment of the RCA, indicating more severe cardiac motion and angulation, a classic risk factor for metal fatigue , 64. AddHowever, there are some contradictions between our results and previous consensus. The indeterminate definition of CSF, stent type, study participants, incompleteness of follow-up and other factors in the included studies may account for the negative results of the meta-regression analysis of the association of follow-up modes, stent length and lesion type with the incidence of CSF.Overall, our study findings support that the incidence of CSF has increased in recent years and that CSF remains an inevitable issue. Accordingly, it is vital for clinicians to be aware of the incidence and predictors of CSF. More importantly, it is useful to learn about the characteristics of stents for proper use of current DESs based on lesion characteristics, and an appropriate detection mode according to the complexity of the lesion is also essential. In addition, it seems unnecessary to carry out longer follow-ups to detect the occurrence of CSF.This study has several limitations. First, studies including only participants with ST or ISR were excluded for their high heterogeneity, which may result in an underestimate of the incidence of CSF among all patients treated with stents, as CSF occurs more frequently among participants with ST or ISR. Second, there were few studies investigating the occurrence of CSF at the stent level, and the funnel plots showed an asymmetrical trend, which may indicate publication bias or be a result of small-study effects, that is, the tendency for smaller studies to show larger treatment effects. Third, even though we tried to account for the high heterogeneity with an inverse variance (DerSimonian–Laird) random effects model, it is difficult to interpret the results. Fourthly, analyses to assess the competing risk were not performed, meaning that the occurrence of the event could have been impacted by competing events. Finally, standards of diagnostic criteria for CSF were not established, and not all information was available, possibly leading to an underestimation of the incidence of CSF. Consequently, large prospective randomized trials are needed to better analyze and understand the phenomenon.CSF is a common phenomenon in patients treated with DES, and the incidence of CSF has increased in the past two decades. This meta-analysis provided reliable estimates of the incidence of CSF among patients with coronary heart disease treated with stent implantation. The point incidence of CSF was found to be 5.5% among patients, 4.8% among lesions, and 4.9% among stents, and the data had high heterogeneity. The results indicated that the duration of stent placement after stent implantation had no impact on the incidence of CSF. In addition, DES type and RCA lesions influenced the incidence of CSF. The potential mechanisms are multifactorial. It is important to understand the relevant features of stents and select appropriate stent types based on the type of lesion. Further research to design appropriate strategies and protocols for the monitoring, management, and prevention of CSF should be a matter of thorough investigation.All datasets analyzed for this study are included in the article/YC, DL, and RY conceived the study design and wrote the manuscript. YC, HL, XY, and JD performed the data collection and analyses. All authors read and approved the final manuscript.


To highlight the current global experience with DCD heart transplantation and explore the evolution of, and compare preservation strategies; examine early clinical outcomes, and discuss the growing use of DCD donors as a new frontier in heart transplantation.The two strategies of DCD heart preservation include NMP using the OCS Heart and TA-NRP followed by either: NMP or CSS. Better understanding the limits of cold ischaemia following TA-NRP will aid in distant procurement. Asystolic warm ischaemia plays an important role in determining immediate post-operative graft function and potential need for mechanical support. Large volume DCD heart transplant units show no difference in survival between DCD and DBD donor heart transplants.In a previously non-utilised source of donor hearts, often viewed as an “unknown frontier” in heart transplantation, DCD hearts are a suitable alternative to brain-dead donor hearts and are likely to remain a permanent part of the heart transplantation landscape. Global uptake is currently increasing, and as understanding of preservation strategies and tolerable ischaemic times improve, utilisation of DCD hearts will continue to grow. Heart failure represents a significant disease burden on healthcare systems worldwide—patients with end-stage heart failure (ESHF), refractory to maximal medical therapy, face frequent hospitalisation, and high morbidity and mortality . In thisGiven these trends, increasing the number of donor hearts sourced is of great interest and importance in order for supply to meet demand. With the majority of heart transplants traditionally being performed from donors after brain death (DBD), donors from a donation after circulatory death (DCD) pathway are emerging as a key alternative source of donor hearts ••, 8••.With heart transplantation from DCD donors still in its relative infancy, in this review, we aim to explore the evolution of preservation strategies that have allowed an increasing uptake of DCD donors as a source of donor hearts, examine early global clinical outcomes, and highlight the growing role and use of DCD donors as a new frontier in heart transplantation.Inspired by surgical and preservation techniques pioneered by Dr. Norman Shumway and his team , the firAlthough the recipient ultimately succumbed to pneumonia and acute rejection after 18 days, the foundation had been laid for future heart transplants to follow . SubsequDespite the world’s first heart transplant being derived from a DCD donor, over the next four decades, donor hearts were almost entirely retrieved from BD donors and were preserved in cold-static storage (CSS) . The relOver time however, uptake of DCD donation for other organs increased with acceptable post-transplant outcomes. Transplantation of kidneys retrieved from DCD donors showed no difference in survival compared to those retrieved from BD donors  and gradWith growing global interest into DCD donors as a means to increase the donor pool, pre-clinical research conducted by our unit at the Victor Chang Cardiac Research institute in Sydney laid the groundwork for our unit to commence a clinical DCD heart transplant program at St. Vincent’s Hospital in 2014. In a porcine DCD model, hearts subjected to 30 min of total warm ischaemia from the time of withdrawal of life support (WLS) followed by administration of a supplemented Celsior cardioplegic solution were able to demonstrate functional recovery during normothermic perfusion on an ex situ working heart apparatus (after 1 h of Langendorff perfusion) [The introduction of the TransMedics  Organ Care System (OCS) Heart normothermic machine perfusion (NMP) device as a means of reperfusing donor hearts with normothermic, oxygenated blood in a resting (or Langendorff) fashion played a vital role in the translation of our pre-clinical findings into a clinical DCD heart transplant program . The ProThe use of NMP allowed, for the first time, an assessment of donor heart viability ex situ—the lack of which had previously inhibited the uptake of DCD donor hearts. Pre-clinical work by our unit demonstrated NMP with the OCS Heart to be superior to CSS in a porcine DCD transplant model . On the Following our initial experience, the uptake of DCD donor hearts began to occur globally. In addition to using the OCS Heart, the Royal Papworth Hospital in the UK also began to retrieve DCD hearts using thoraco-abdominal machine perfusion (TA-NRP) ••, 25. TDCD donation processes involve the withdrawal of life support (WLS). In DCD donors where the heart is being retrieved, WLS has generally occurred in a controlled environment such as in an intensive care unit or anaesthetic bay (Maastricht Category III donors). Occasionally , retrieval of the donor heart and other organs has occurred after controlled WLS in brain-dead donors (Maastricht Category IV donors) ••, 31. OIn Australia, death determined by circulatory criteria is defined as irreversible cessation of the circulation. Based on this definition, re-establishment of the circulation by normothermic regional perfusion is not permitted after death has been determined by DCD pathway. For this reason, we developed a direct procurement protocol (DPP) for retrieving hearts from DCD donors. Our retrieval team waits up to 90 min following the WLS for the donor systolic blood pressure (SBP) to fall below 90 mmHg ••. Once Total warm ischaemic time (WIT) is the duration between WLS and administration of cardioplegia at the time of organ procurement. Different units have varying definitions for the functional warm ischaemic time (fWIT). In our unit, this is duration between SBP < 90 mmHg and cardioplegia. We refer to the period of time from asystole to the administration of cardioplegia as the asystolic warm ischaemic time (aWIT). Several measures have been adopted to minimise the fWIT. One measure that has been implemented in Spain, Belgium, and certain USA units has been to undertake WLS with the donor prepped and draped in the operating room prior to WLS negating the accumulation of WIT during transport ••, 30••.Two main strategies currently assist in assessing viability of DCD donor hearts: normothermic machine perfusion (utilising the OCS Heart) and normothermic thoraco-abdominal regional perfusion.NMP is the method of reperfusion in Australia for DCD donor hearts as TA-NRP is not allowed. In regions where TA-NRP is not possible, NMP is also used in the UK ••. The hIn high-risk donors in whom pre-retrieval coronary angiography may not be possible in certain locations either due to policy or availability of hospital resources, the OCS Heart can safely facilitate ex situ coronary angiography upon return to the recipient hospital in order to exclude coronary disease . HaemodyThe true meaning of lactate profiles in the setting of NMP is yet to reach a consensus; initially thought to be a predictor of graft failure, the initial experience with the OCS Heart had clinicians aiming for a lactate level of < 5 mmol/L to determine if hearts were suitable for transplant , 36. MorTo date in Australia, 77 DCD hearts have been successfully transplanted using the OCS Heart . The appafter the donor has been declared dead [TA-NRP is an alternative to NMP as a means for assessing DCD donor heart viability through an in situ assessment of DCD donor hearts. It was initially pioneered in donor DCD hearts destined for transplantation by the Royal Papworth Hospital group as a means of providing a functional and structural assessment of the donor heart without having to rely on OCS Heart lactate profiles, which were a point of conjecture ••, 40. Tred dead ••, 40. Bred dead ••, 40.The surgical approach to TA-NRP varies between countries in regard to the timing of interventions during the donation process. As antemortem cannulation and heparinisation is not permitted in the UK, once the mandatory 5-min “stand-off” period is observed, the Papworth technique employs a central cannulation strategy following median sternotomy in order to establish bypass with heparin being administered directly into the heart ••, 40. TThe Papworth group in the UK has the largest reported experience to date with TA-NRP with 22 DCD hearts being retrieved following TA-NRP assessment of organ viability ••. ThreeThe Vanderbilt University Medical Center, Nashville, TN, has published to date the largest known cohort of DCD donor hearts assessed through TA-NRP and then preserved with CSS ••. ResulGoing forward, as more centres adopt TA-NRP/CSS and potentially foray into distant procurement, the role extended CITs play on eventual graft function will be of particular interest in determining the optimal transport strategy between CSS and NMP. The Belgium experience of the first paediatric heart transplant from a distantly procured DCD donor using TA-NRP/CSS reports a cold ischaemic time of 117 min  and reprp = 0.002). For the purposes of our protocol, we define “cold ischaemic time” as the time taken from the delivery of cardioplegia during donor heart procurement until the time of reperfusion on the OCS Heart. Cold ischaemic time was not associated with an increased risk of ECMO.In the St. Vincent’s initial experience with DCD heart transplantation, we noted an ECMO rate of 35% for PGD in the first 23 patients who were transplanted with DCD hearts following OCS Heart NMP ••. DurinSince that initial series, our program has grown and we have been able to learn from our experiences along the way. The addition of tirofiban to our blood collection protocol during the DPP in response to module filter clotting issues has resulted in the subsequent absence of module filter clotting and is an example of one of the tweaks successfully made during our close to 9-year experience with DCD heart retrieval . FurtherWhilst our initial ECMO rate was high, our ongoing use of the OCS Heart has enabled ongoing familiarisation with DPP techniques and an appreciation of the need to minimise aWIT. The ECMO rate in the subsequent 54 DCD heart transplants that have occurred since the initial series is now at 7% . DespiteThe future approach to DCD hearts should be to consider ways to minimise aWIT in particular, or to develop strategies of allograft preservation aimed at mitigating potential cellular damage that may occur during aWIT.The implementation of a DCD heart transplantation program has been shown to have a tangible impact on heart transplant wait lists. Messer et al. •• reportStarting a DCD clinical heart transplantation program requires significant resources, coordination, and training. Utilising normothermic machine perfusion involves logistical and financial consideration into the transportation and constant monitoring of the OCS Heart. In contrast, TA-NRP requires the initiation of cardiopulmonary bypass and the management of a heart–lung bypass machine and oxygenator.Adopting either TA-NRP or NMP is a decision made based on the specific needs of the transplant unit, local laws, and the geographical areas serviced. In Australia for example, due to the size of the country, a DPP followed by NMP with the OCS Heart is advantageous in facilitating distant procurement to interstate and rural retrieval locations ••, 23.The administration of antemortem heparin in DCD donors is an ethically challenging subject, with no clear international consensus , 44. TheFollowing withdrawal of life support, the physiological milieu is one of profound acidosis; data from our DCD retrieval experience demonstrates mean pre-withdrawal pH to be 7.41 (± 0.06), and this drops to a mean pH of 7.10 (± 0.23) following asystole ••. This Acid Sensing Ion Channel 1a (ASIC1a) plays key roles in mediating cellular injury in response to acidosis , 48. A pData from two of the longest and largest DCD heart transplantation programs in Australia and the UK have demonstrated that survival from DCD heart transplantation is equivalent to traditional DBD donors. DCD programs are now emerging across Europe and the USA with early success. It is now established that the uptake of DCD donor hearts has the impact of improving overall heart transplantation numbers and can legitimately contribute to the reduction of global transplant wait lists. The decision to choose between NMP and TA-NRP retrieval strategies is multifactorial and location dependent. Ongoing research and clinical experience into pushing the boundaries of preservation strategies will provide the impetus for the ongoing evolution of DCD heart transplantation.


Neuroinflammation is one of the common features in most neurological diseases including multiple sclerosis (MScl) and neurodegenerative diseases such as Alzheimer’s disease (AD). It is associated with local brain inflammation, microglial activation, and infiltration of peripheral immune cells into cerebrospinal fluid (CSF) and the central nervous system (CNS). It has been shown that the diversity of phenotypic changes in monocytes in CSF relates to neuroinflammation. It remains to be investigated whether these phenotypic changes are associated with functional or metabolic alteration, which may give a hint to their function or changes in cell states, e.g., cell activation. In this article, we investigate whether major metabolic pathways of blood monocytes alter after exposure to CSF of healthy individuals or patients with AD or MScl. Our findings show a significant alteration of the metabolism of monocytes treated with CSF from patients and healthy donors, including higher production of citric acid and glutamine, suggesting a more active glycolysis and tricarboxylic acid (TCA) cycle and reduced production of glycine and serine. These alterations suggest metabolic reprogramming of monocytes, possibly related to the change of compartment (from blood to CSF) and/or disease-related. Moreover, the levels of serine differ between AD and MScl, suggesting different phenotypic alterations between diseases. Neurodegenerative and inflammatory diseases such as Alzheimer’s disease (AD) and multiple sclerosis (MScl) are continuously gaining focus from the scientific and public audience due to the high incidence and devastating consequences in terms of life quality and care-need of patients. Although the course, symptoms, causes, and characteristics of these neurological diseases are highly heterogeneous, some aspects are in common, i.e., the presence of neuroinflammatory features. Both diseases are characterized by microglial activation ,2,3,4, iOne of the hypotheses is that once the monocytes enter the CSF through the choroid plexus (CP), they undergo phenotypic changes and take part in the inflammatory response to eventually help the resolution of the innate inflammation ,22. The 13C-glucose tracing) increases confidence in identifying the metabolites affected by the study conditions or experimental manipulations.To have a more complete overview of the changes that CSF-infiltrating monocytes undergo, it is necessary to also consider immunometabolism and its alterations. Lately, it has been pointed out as fundamental in the regulation between pro- and anti-inflammatory profiles . The metIn our previous study , we char13C2-glucose tracing experiment [13C pyruvate to 13C lactate and a reduced production of serine [We also showed, using the previously validated 1,2-periment , how thef serine .13C2 glucose tracing experiment on monocytes targeting key-role metabolites of the major metabolic pathways of the cell  cycle, lactate production from pyruvate, and glutamine/glutamic acid metabolism). Intracellular and secreted unlabeled and 13C glucose-derived metabolites were identified and quantified using HPLC-MS/MS. In this study, we identified the rewiring of the glucose metabolism of monocytes after CSF treatment, and the differential metabolic alterations in neuroinflammatory conditions such as MScl (MScl-CSF treatment) in comparison with neurodegeneration conditions such as AD (AD-CSF treatment).In this study, we aim to comparably evaluate the overall metabolism of monocytes in CON and AD individuals in comparison to MScl with more pronounced neuroinflammation. We performed a 1,2-This study was registered and approved by the Ethics Commission of Charité—Universitätsmedizin Berlin , Berlin, Germany.PBMCs were obtained from the German Red Cross and stored at −80 °C until incubation. Monocytes were then isolated from PBMCs with MACS (Pan Monocyte Isolation Kit (human), Miltenyi Biotec, Bergisch Gladbach, Germany).After isolation, monocytes were incubated in a 24-well/plate in four different conditions with three biological replicates for each group: medium only without stimulation (3 × NS), with the addition of 30% in volume of CSF of healthy individuals (3 × CON), with the addition of 30% in volume of CSF from AD patients (3 × AD), and with the addition of 30% in volume of CSF of MScl patients (3 × MScl). Before use, pH values of all CSF samples were measured. Only CSF with a pH of about 7 was used in this study.2, in a medium without glucose, pyruvate, glutamine, and phenol red  added with labeled glucose  to a final concentration of 4.5 g/L, and with 10% FBS . As the incubation of monocytes in a medium without pyruvate, glutamine, and phenol red may be challenging, the well-being of the cells needs to be monitored with a microscope at regular time intervals.The monocytes were incubated for 5 h at 37 °C, with 5% CODetails of isolation and culture of monocytes are described elsewhere .g. Then, the supernatant  was separated from the cell pellet and centrifuged at 15,000× g, for 10 min at 4 °C before the analysis with HPLC-MS/MS.After the incubation, the cell suspension was transferred to Eppendorf tubes and centrifuged for 10 min at 4 °C at 300× 2O: acetonitrile (ACN)  were added for the extraction. After thorough vortexing and incubation on ice for 5 min, the samples were centrifuged at 15,000× g for 10 min at 4 °C. Aliquots of 75 µL of the supernatant without disturbing the cell remainder were used to obtain the cell extract (representing an intracellular level of metabolites). Samples were stored at −80 °C until the HPLC-MS/MS analysis.After shock-freezing of the cell pellets using liquid nitrogen 100 µL of HDetails of monocyte extraction and sample preparation are previously described in detail .The chromatographic conditions, the MS/MS parameters, and the dynamic multiple reaction monitoring (dMRM) method were optimized and validated. Details of the analytical method are reported previously .2O:ACN (1:1).Standard solutions for calibration levels and quality controls (QCs) were prepared in serial dilution from stock solution (1 mg/mL) in Milli-Q H3PO4 in ACN:H2O (9:1). Mobile phase A was 10 mM CH3COONH4 in H2O with the addition of InfinityLab deactivator additive . Mobile phase B was 10 mM CH3COONH4 in ACN with the addition of InfinityLab deactivator additive (Agilent Technologies). We added 1 mL of additive for 1 L of mobile phase. HPLC separation was achieved by running a linear gradient from 10% to 40% to 10% mobile phase A in 20 min. The optimized dynamic multiple reaction monitoring (dMRM) was conducted by applying both positive and negative ionization mode.Analyses of samples, QC, and calibration levels were conducted with an Agilent 1290 Infinity II HPLC system coupled with an Agilent 6495 QqQ mass spectrometer (MS/MS) with an Agilent jet stream source with electrospray ionization (AJS-ESI), both controlled by MassHunter 10 Data Acquisition software . For the separation of the metabolites, an Agilent InfinityLab Poroshell 120 HILIC-Z column  was used. To optimize the chromatographic peak shape of citric acid and phosphates, passivation of the system was needed with an overnight wash of the system with 0.5% HFor further specifications on HPLC conditions, MS parameters, and mobile phase preparation, refer to Giacomello et al. .Chromatographic and spectrometric results were evaluated with MassHunter 10 Quantitative Analysis program G3336 . For graphical summaries, Prism 7 (GraphPad) was used.For the statistical analysis, OriginPro 2021b (OriginLab) was used. Normal distribution and equality of variances were evaluated with Shapiro–Wilk test and Levene’s test, respectively. For the evaluation of the significant variation between groups, a one-way ANOVA was conducted for normally distributed groups and Kruskal–Wallis for those not normally distributed. To determine which group was significantly different, a Tukey’s test or a Dunn’s test (non-parametric) was conducted.13C2 pyruvate, 1,2-13C2 lactate, 2-13C glycine, and 1,2-13C2 glutamine produced by human primary monocytes, incubated in vitro without stimulation or with the addition of CSF of healthy donors or patients with AD or MScl, were successfully quantified. Of note, the concentrations of 2,3-13C2 serine and 1,2-13C2 citric acid were lower than the limit of quantitation (LOQ), and thus they were considered detectable but not quantified metabolites. This confirmed the integration and conversion of the supplemented 1,2˗13C2 glucose into the glucose metabolism of the primary monocytes. ATP, AMP, acetyl coenzyme A (AcCoA), fructose-6-phosphate, glyceraldehyde-3-phosphate, phosphoglyceric acid, and ribose-5-phosphate concentrations were below the limit of detection (LOD) under our in vitro experimental conditions. These results are summarized in Using our validated method , unlabelAs common in HILIC separation ,46,47 anp = 0.0073), glutamine (p = 0.0004), and pyruvate (p = 0.0387) were detected in monocytes after treatment with CSF  in comparison to non-stimulated (NS) monocytes. In contrast, serine (p = 0.0356) and glycine (p = 0.0166) were quantified at a lower concentration compared to the NS group. These observations were partly confirmed with post hoc tests , i.e., the concentrations of intracellular citric acid and glutamine were significantly increased in monocytes after exposure to CON-CSF, AD-CSF, and MScl-CSF in comparison with the NS group, whereas pyruvate concentration was significantly increased only in the CON-CSF-treated group compared with NS. Intracellular serine and glycine concentrations were finally significantly different only between AD-CSF-treated and MScl-CSF-treated monocytes, each compared to the NS group.As shown in 13C2 glucose-derived compounds were 2,3-13C2 pyruvate and 1,2-13C2 lactate. A small amount of 13C-glutamine was quantified in CSF-treated monocytes (>LOQ), while its concentration was below LOQ in untreated monocytes , glutamine (p = 9.0 × 10−7), pyruvate (p = 6.9 × 10−5), serine (p = 1.5 × 10−10), and glycine (p = 1.3 × 10−6) were detected between the NS and the CSF-treatment groups (p = 4.6 × 10−5) shows significant variations in concentrations only between NS and CSF of patients (AD and MScl). There are also some significant differences between CSF-treated groups. Lactate (p = 0.0125), serine, and glutamic acid displayed a significant variation between CON and AD, whereas lactate, glutamine, and glutamic acid differed between CON and MScl and glutamine, and serine between AD and MScl , significant differences in the level of citric acid  and lactate , and only in small amounts to glutamine  and glycine (2-13C glycine), as shown in 13C2 serine and 1,2-13C2 citric acid were detected in all samples, but the concentrations were lower than LOQ, and thus they are not shown in 13C2 glutamine could only be quantified in the monocytes treated with CSF and 2-13C glycine only in the NS group. Differential abundances of 2,3-13C2 pyruvate and 1,2-13C2 lactate were significantly increased in CSF-treated monocytes .Determination of the incorporation of p-values obtained with the one-way ANOVA  or the Kruskal–Wallis test (non-parametric) and the results of the Tukey’s or Dunn’s tests to establish the differences between each group.To summarize the significant differences between all groups, 13C-labeled) during the incubation. The concentration of glucose in CSF of healthy individuals or patients with AD or MScl is reported to be similar [We took into consideration that there are some naturally abundant metabolites in CSF, e.g., lactate, pyruvate, and glutamine. These compounds were also detected and quantified. In  similar ,49,50,5113C from labeled glucose into its metabolites allows insights into the metabolic pathway that was preferentially taken during in vitro stimulation. Further to small amounts of the remaining endogenous glucose present in the monocytes or the CSF, the only source of glucose was 1,2-13C2-glucose added to the glucose-free medium. Primary human monocytes isolated from the peripheral blood of healthy individuals were used in this study. The main limitation of our study was the number of monocytes obtained from a limited volume of the peripheral blood, which in turn resulted in low amounts of detected metabolites. However, the feasibility to apply this method to study changes in cellular metabolism  in a small sample of human primary cells was demonstrated. Although not all glucose-derived metabolites could be detected due to the limitation mentioned above, an increased level of both unlabeled and/or labeled citric acid, glutamine, and pyruvate in CSF-treated monocytes and in the culture medium was consistently detected, whereas serine and glycine were consistently reduced after treatment with CSF. Our findings suggest the rewiring of the glucose metabolic pathway in monocytes after treatment with CSF, possibly due to an increased cellular activation. Commonly, activation of human innate immunity requires alteration of cellular metabolic pathways largely to favor glucose metabolism [The incorporation of tabolism ,54,55,5613C2 citric acid are displayed in neither The significant increase in the citric acid concentration, once monocytes came into contact with CSF no matter from which group, follows the increase in pyruvate mentioned above, suggesting a more active tricarboxylic acid (TCA) cycle as well. Due to the limited amount of labeled pyruvate, and the preferential pathway towards the formation of lactate, it is not a surprise that other labeled compounds of the TCA cycle were not quantifiable. Moreover, rate-limiting in the TCA cycle is the citrate synthase catalyzed step . As labeAs highlighted in the introduction, monocytes undergo phenotypic changes and contribute to the progress of inflammation. As this requires more energy, metabolic reprogramming is observed. The results shown in this study corroborate this hypothesis. In line with another study by Ren et al. , monocyt13C-labeled) in monocytes treated with CSF confirms an enhancement of the TCA cycle in CSF-treated monocytes. However, this amino acid is also present in CSF. Thus, it serves as an extra source of glutamine that is not present in the NS group, which was incubated in a glutamine-free medium. However, there is a clear increase in glutamine amounts in cell extract and medium compared with CSF only , especially for healthy individuals and, to a slightly lesser extent, for patients with lower neuroinflammatory conditions (AD). This also implies the need for energy  during phenotypic alteration and a differentiation in the metabolism rewiring between AD and MScl neuroinflammatory conditions. Furthermore, glutamate is neurotoxic at certain concentrations in CSF, although it is essential for the homeostasis of the body. Conversion of glutamate to glutamine is one of the mechanisms to eliminate the excess of glutamate to maintain system homeostasis. Of note, MScl patients usually show elevated levels of glutamate in the brain, leading to excitotoxicity ,66,67. HThe CSF-stimulated groups exhibited lower concentrations of glycine and serine in comparison with NS. The pathway towards the production of these amino acids passes through 3-phosphoglyceric acid (3PG). Thus, monocytes, when stimulated with CSF, diverge the metabolism of 3PG towards the production of pyruvate instead of glycine and serine. Under LPS-stimulation, glycine shows modulatory effects on cytokine production of monocytes, that is, a reduction in TNF-α and IL-1β expression . These rIn summary, we report a significant alteration in the metabolism of monocytes when incubated with CSF. Overall, the metabolism showed a preference towards the TCA cycle and production of glutamine for the cells incubated with CSF, especially those from patients with AD and MScl. Other metabolic pathways such as serine and glycine production were down-regulated. These changes in glycolysis imply the alteration of monocyte phenotypes towards a reactive state. However, our study has some limitations, including: (1) it presents a limited number of biological replicates due to the rarity of the specimen CSF considered; (2) it uses an in vitro experimental model that may not completely explain the in vivo situation, and; (3) due to low cell numbers, some concentrations of the metabolites were lower than the limit of detection and/or limit of quantitation. Therefore, optimization and further development of the analytical methods are required, including the use of labeled glutamine for the parallel characterization of TCA cycle metabolites ,72 and tUnderstanding how blood monocytes respond to the local environment  at both phenotypic and functional/metabolic states will provide further insights on how to better regulate these cells, especially in neurological diseases, in which these cells play an important role ,74.


The area under the receiver operating characteristic curve (AUROC) for using the Nakagami parameter to detect MEE was 0.90, and the sensitivity, specificity, and accuracy were 82.5%, 97.5%, and 79.6%, respectively. The Nakagami parameter for tympanogram types B/C was higher than that for tympanogram type A (p < 0.05); it was also higher in the subjects with hearing loss (p < 0.05). Quantitative transmastoid ultrasound based on the Nakagami parameter analysis has the potential to detect MEE and evaluate hearing loss.Medical history taking, otoscopy, tympanometry, and audiometry are clinical methods to diagnose middle ear effusion (MEE); however, these procedures are experience-dependent and result in misdiagnosis under unfavorable conditions of the external auditory canal in non-cooperative young children. This study aimed to explore the use of transmastoid ultrasound combined with the Nakagami parameter analysis to detect MEE in children aged 3–5 years and to compare the proposed method with clinical evaluation methods. A total of forty subjects were enrolled; for each subject, a single-element ultrasound transducer of 2.25 MHz was used to measure backscattered signals returned from the mastoid for estimating the Nakagami parameter, which is a measure of the echo amplitude distribution. Tympanogram and hearing loss were also measured for comparisons. The results showed that the Nakagami parameter in the patients with MEE was significantly larger than that of the normal group ( Acute otitis media (AOM) usually presents with otalgia, restless sleep, and hearing impairment, and it is the second most common cause for children to visit the pediatrician . AOM is According to the clinical practice guidelines, pneumatic otoscopy is strongly recommended for diagnosing MEE in children. For young children, a relatively narrow ear canal and impacted cerumen may interfere with the otoscope’s ability to inspect the condition of the tympanic membrane, leading to misdiagnosis. Tympanometry is another method recommended to assist in the diagnosis of MEE, which usually presents as type B  or C . When a child has MEE, audiometry is an important tool for assessing hearing sensitivity. The average hearing loss in children with MEE is 27 dB . MEE thaThe mastoid cavity is an air-filled space in a bony structure, which is connected to the middle ear cavity and is influenced by MEE. It has been confirmed that mastoid effusion (ME) influences the intensity of ultrasound signals backscattered from the microstructures when ultrasound is transmitted into the mastoid . Our preThe aim of this study is to explore the diagnostic performance of using the Nakagami parameter in detecting MEE in patients aged 3–5 years. In the Materials and Methods section, we explain how we enrolled subjects, conducted measurement procedures, and analyzed data. The Results section reports the Nakagami parameters of the mastoid for comparisons with the findings of MEE history, tympanogram type, and hearing loss for discussion. The Discussion section compares the results with some existing works, interprets underlying mechanism of change in the Nakagami parameter, and addresses the significance and limitations of this study. The Conclusions section indicates the main contribution of this study; that is, transmastoid ultrasound using Nakagami parameter analysis can detect MEE in children as well as distinguish between tympanogram types and subjects with and without hearing loss, offering a solution for routine examinations and follow-ups to children with potential AOM.This study was approved by the Joint Institutional Review Board of Chang Gung Memorial Hospital (No. 201600985B0). Children were presented to our outpatient department with ear discomfort or suspected otitis media as reported by their families from January 2018 to December 2018. A total of forty participants aged 3–5 years were enrolled in this study. Informed consent was obtained from each patient and their relatives.A total of 20 healthy volunteers without any medical histories in MEE-related problems participated in the normal group (40 ears), and another 20 subjects that had been diagnosed as having bilateral MEE and scheduled for being undergoing grommet surgery to drain effusion were enrolled in the MEE group (40 ears). All subjects were re-examined through otoscopy by an experienced otolaryngologist; after that, their ear canals were sealed using a 226 Hz probe tone for tympanometry measurements by professional audiologists. Subsequently, audiologic evaluations by pure tone audiometry (PTA) were performed. Interacting with children who are undergoing clinical examinations can be very challenging, and thus conditioned play audiometry was used to improve cooperation. Air conduction and bone conduction hearing levels at 0.5, 1, 2, and 4 kHz were recorded because the speech spectrum typically falls into the range between 500 to 4000 Hz. Children in the normal group were confirmed to have tympanogram type A and normal otoscopic findings; those with the confirmed diagnosis of MEE were identified following otoscopic findings in accompanying with tympanogram type B/C and surgical findings fed back from the surgical team. The sample size was determined by the software G*power  (versionE(·) is the statistical mean operator, and R is the envelope signal. The probability density function f(r) of the Nakagami distribution for the envelope signal r is given by (Equation (2))Γ(·) and U(·) are the gamma function and the unit step function, respectively. The scaling parameter Ω is related to echo energy. The Nakagami parameter is a shape parameter of the Nakagami distribution, and it is able to describe various scattering conditions in the tissue. Values of the Nakagami parameter <1 means that the echo amplitude distribution is the pre-Rayleigh distribution, while a value = 1 indicates the Rayleigh distribution for the backscattered statistics. In this condition, higher Nakagami parameter values represent that the scattering medium belongs to a more supportive environment for producing echoes. Five independent measurements were carried out to obtain an average Nakagami parameter for each subject.Transmastoid ultrasound examination of each subject was performed using an ultrasound system and the algorithm proposed in a previous report . The syst-test; p value < 0.05 was considered a statistically significant difference. To evaluate the diagnostic value of the Nakagami parameter in determining the presence of MEE, the receiver operating characteristic (ROC) curve analysis at 95% confidence interval (CI) was performed to obtain the area under the ROC (AUROC). The sensitivity, specificity, and accuracy were also reported. Nonparametric tests were used for small sample size data. Additionally, the data in the normal control and MEE groups were further regrouped to explore the effects of MEE duration, tympanogram type, and hearing loss on the Nakagami parameter of the mastoid. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 22.0 .Typical backscattered signals measured from the normal control and the MEE groups were plotted. The Nakagami parameters of the two groups were expressed as median and interquartile range (IQR) and compared using the independent p = 0.65 and p = 0.74, respectively).Twenty children without MEE  as the normal group and 20 children with MEE  as the MEE group were enrolled. There were no significant differences in the age and sex between the groups (p < 0.001), indicating that the echo amplitude distribution of the mastoid tends to vary toward the Rayleigh statistics when the middle ear is filled with effusion. As shown in The statistical properties of ultrasound backscattered signals were further quantified by estimating the Nakagami parameter. The median of the Nakagami parameter for the MEE group was 0.39 (IQR: 0.38–0.40), which was larger than 0.35 (IQR: 0.34–0.36) for the normal group, as shown in p < 0.05) and between the normal and MEE >3 months (p < 0.05).The data of the Nakagami parameter in the normal control and MEE groups were further classified by the MEE duration of <3 months and >3 months, as shown in p < 0.05) and between the types A and C (p < 0.05). The Nakagami parameters corresponding to different types of tympanograms were compared, as shown in p < 0.05). However, the Nakagami parameter was independent of the level of hearing loss although it was able to discriminate between the normal control and the subjects with hearing loss.The data of the Nakagami parameter in the MEE group were compared according to the levels of hearing loss ≤30 dB and >30dB, as shown in Standard diagnostic tools of MEE, such as pneumatic otoscopy, which is considered a visual assessment method, are often unreliable if patients are uncooperative. The diagnostic accuracy of traditional otoscopy for MEE detection varies between 40 and 70% . The diaBesides MEE detection, we also explored the dependencies of the Nakagami parameter on MEE history, tympanogram type, and hearing loss. Patients with otitis media for >3 months are at risk of hearing loss , which iConsidering another risk factor for ventilation tube insertion, hearing loss >30 dB for the speech frequencies (300 to 3000 Hz) is unacceptable for conversational communication  because This study has some limitations. First, the number of subjects is insufficient, and the middle ear cavity individually varies; thus, a large-scale, randomized control study is necessary to explore considerations in practical uses prior to using quantitative transmastoid ultrasound as a reliable tool to evaluate MEE in a clinical setting. Second, there are multiple factors, including volume, fluid viscosity, mucosal thickness, E tube dysfunction, and bacterial toxins, that influence the hearing of patients. The dependencies of the Nakagami parameter on the above factors should be further clarified to specifically confirm the role and position of quantitative transmastoid ultrasound in MEE detection. Third, children aged under 3 years were not enrolled in the study. More pediatric cases need to be included and validated. Finally, the statistical properties of ultrasound backscattered data may differ between different systems. Developing a system dedicated to transmastoid ultrasound measurement and an optimized computational algorithm is a key future work.We have explored the diagnostic performance of using quantitative transmastoid ultrasound based on the Nakagami parameter to detect MEE in children aged 3–5 years. The AUROC was 0.90, and the sensitivity, specificity, and accuracy were 82.5%, 97.5%, and 79.6%, respectively. It demonstrated that the underlying mechanism that MEE produces mastoid effusion to further affect the statistical properties of ultrasound backscattered signals measured from bony microstructures can be applicable to children, endowing the Nakagami parameter of the mastoid with the ability to detect pediatric MEE. In addition, the Nakagami parameter of the mastoid also had the ability to discriminate between tympanogram types A and B/C and between the subjects without and with hearing loss, but it was unable to identify how long the MEE history had been due to the lack of sensitivity in tissue characterization with low-frequency ultrasound. In the future, transmastoid ultrasound combined with the Nakagami parameter can be used for detecting MEE and evaluating hearing loss, providing routine examinations and follow-ups to children with potential AOM.


Biomarkers in CSF could provide etiological clues and diagnostic tools for psychotic disorders. However, an overview of all CSF findings in individuals with psychotic disorders compared to healthy controls is lacking.To analyse CSF findings from individuals with psychotic disorders compared to healthy controls.PubMed, EMBASE, Cochrane Library, Web of Science, ClinicalTrials.gov, and PsycINFO were searched November 3rd, 2021. Case-control studies including patients with non-affective, psychotic disorder compared to healthy controls measuring at least one biomarker in CSF are included. Standardized Mean Differences (SMD) and random-effects analyses were used.141 studies, covering 192 biomarkers, were included. 161 biomarkers have not previously been included in meta-analyses. Most markers measured showed no significant differences, including the dopamine metabolites HVA and DOPAC. Patients with psychotic disorders showed increased CSF levels of noradrenaline , MHPG , 5-HIAA , kynurenic acid , kynurenine , IL-6 , IL-8 , anandamide , albumin ratio , total protein , and glucose . Neurotensin  and GABA  were decreased.These findings suggest that dysregulation of the immune and adrenergic system and blood-brain barrier dysfunction might play a role in the pathophysiology of psychotic disorders.No significant relationships.


We present a surgical technique combining arthroscopic-assisted lower trapezius tendon (LTT) transfer with autologous semitendinosus tendon and long head of biceps tendon (LHBT) superior capsule reconstruction (SCR) for massive irreparable posterosuperior rotator cuff tears. The patients are placed in the beach-chair position with the ipsilateral lower leg prepared simultaneously. After both tendons are harvested, 1 limb of a semitendinosus graft is fixed with the LTT via a Krakow suture. The LHBT is then fixed by an anchor 5 to 8 mm posterior to the bicipital groove and tenotomized distally. The transverse humeral ligament is released afterward to provide better visualization. A Beath pin is introduced from anterolateral portal, aiming at the bicipital groove, and drilled posteriorly until it exits at the infraspinatus footprint. Next, 4.5- and 8-mm cannulated drills are used sequentially to create a humeral tunnel. A shuttle suture passed through infraspinatus fascia in the back brings the EndoButton and looped semitendinosus graft from posterior to anterior of the humerus, until the EndoButton flips and is fixed inside the bicipital groove. The shoulder is placed in 45° abduction and 30° external rotation. The free limb of semitendinosus tendon is then sutured with LTT with the desired tension. The whole procedure of the surgery. This is a 60-year-old male heavy worker who worked at a construction area with preoperative pseudoparesis. Active forward elevation was about 80°. He also had external rotation lag and positive Hornblower sign. Active internal rotation was limited to the sacrum. No osteoarthritis was seen on the plain x-ray, but a massive posterosuperior cuff tear with retraction to glenoid level was observed. Both supraspinatus and infraspinatus had Goutallier 3 fatty infiltration. The patient was put in a beach-chair position with ipsilateral knee prepared as well. The semitendinosus tendon was harvested with both ends sutured with no. 2 Ethibond. The LTT was detached, and the tendon part of it was whipstitched to facilitate further manipulation. Viewing from lateral portal, a basic arthroscopy exam was performed to confirm the retractability of the cuff. A suture-based anchor is passed from anterolateral portal and inserted 5-mm posterior to the bicipital groove near the cartilage of the humerus. One loop was passed through the biceps first, and then we used a bird beak to penetrate the biceps. After knot tying, the biceps was rerouted posteriorly. Then we cut the biceps distal to our knots at the entrance of the bicipital groove. The proximal attachment of the biceps on the glenoid side was preserved, providing native fixation, while the lateral part worked as a LHBT SCR. Then the THL was released, providing better visualization for humeral tunnel drilling and graft passage. More tension could be given by penetrating the intra-articular LHBT in a more medial position. A strong spacer effect was achieved. Then we use a Beath pin introduced from anterolateral portal, aiming at the bicipital groove, and drilled posteriorly until it exited at the upper part of the native infraspinatus insertion point. A 4.5-mm cannulated drill was used to ream from anterior to posterior to create a humeral tunnel, and the length of the tunnel was measured. Another 8-mm drill was used to ream from posterior to anterior until the desired length. Then we passed a suture along the infraspinatus muscle, out of the opening of the infraspinatus fascia. The free limb of semitendinosus tendon not fixed with LTT was passed from the loop of a 20-mm EndoButton and worked in a double fashion. The EndoButton was shuttled intra-articularly from posteriorly to anteriorly with the graft until it exited the bicipital groove and flipped for final fixation. The free limb of semitendinosus tendon was then pulled backward until the desired tension, checked intra-articularly. This end was fixed side by side with the LTT with Krakow suture. Additional sutures could be made onto the remnant cuff, providing more soft tissue coverage on top of the humeral head. Although it might be a nonfunctional cuff, we hoped it could provide some additional strength to stabilize the humerus. Postoperative plain x-ray showed better acromiohumeral distance right after the surgery. Six months after the surgery, the patient had about 130° of active forward elevation and acceptable active external rotation without lag sign. The acromiohumeral distance was preserved. The final construct was like this cartoon. The ipAn 8-cm horizontal incision is made just below the spine of the scapula over the lower trapezius tendon insertion. The subcutaneous tissue is dissected, and the underlying fat is removed until the tendon is identified. The LTT is then detached from the scapula spine and mobilized superiorly from the middle trapezius and medially until the medial border of scapula. Care must be taken to avoid injury to the spinal accessory nerve that runs 3 to 4 cm medial to the scapula. The tendon part of LT was whipstitched with no. 2 Ethibond (Ethicon) to facilitate further manipulation A.Fig 2HaThe semitendinosus autograft was harvested full length from the insertion site with a tendon stripper  with both ends sutured with no. 2 Ethibond B. One liViewing from lateral portal, a suture-based anchor is passed from anterolateral portal and inserted 5-8 mm posterior to the bicipital groove near the cartilage of humerus. The surgical techniques are the same as Chiu et al.Viewing from lateral portal, the bicipital groove is identified. A Beath pin (Smith & Nephew Endoscopy) is introduced from anterolateral portal, aiming at bicipital groove A, which After the EndoButton is flipped and fixed at the bicipital groove, the shoulder is placed in 45° abduction and 30° external rotation. The free limb of semitendinosus tendon not yet fixed with LTT can be pulled backward to the desired tension. After adequate tension is achieved and checked intra-articularly under arthroscopy A, this eThe shoulder is protected in the brace for the first 6 weeks. From 6 to 12 weeks, active range of motion is permitted, with avoidance of any activation of the LTT transfer. Three months after the operation, the patients are allowed to perform shoulder abduction and external rotation with scapular retraction.The whole procedure of the surgery is shown in the ,Open LTT transfer has been used to restore external rotation in patients with brachial plexus injuries, with promising results.Regarding the humerus tunnel drilling during the surgery, Valenti and WerthelThe anterior rotator cable is the primary force-transmitting structure at the proximal humerus.


R1-nj phenotype and corrected using the red root marker or using a gold-standard test that uses plant traits. RWS and Mo-17-derived inducers showed higher average induction rates and the commercial dent hybrid background showed higher inducibility. In contrast, sweet corn and flint backgrounds had a relatively lower inducibility, while non-stiff stalk and stiff stalk backgrounds showed intermediate inducibility. For the poor-performing donors (sweet corn and flint), there was no difference in the HIR among the inducers. Anthocyanin inhibitor genes in such donors were assumed to have increased the misclassification rate in the F1 fraction and, hence, result in a lower HIR.Doubled haploid technology is a feasible, fast, and cost-efficient way of producing completely homozygous lines in maize. Many factors contribute to the success of this system including the haploid induction rate (HIR) of inducer lines, the inducibility of donor background, and environmental conditions. Sixteen inducer lines were tested on eight different genetic backgrounds of five categories in different environments for the HIR to determine possible interaction specificity. The HIR was assessed using the  Doubled haploid (DH) technology has become the main method of inbred line development in private and public maize breeding programs which possess the necessary infrastructure for adoption . A signiIn vivo haploid induction in maize is made through intra-specific crosses with genotypes known as haploid inducers . These gR1-nj gene. This gene leads to anthocyanin production in both the scutellum and aleurone layers of seeds, where proper fertilization and central cell development occurred. When inducer chromosomes are excluded from zygotic cells or the egg develops parthenogenetically, haploid embryos with unpigmented scutellum are formed. This difference in scutellum pigmentation allows the differentiation between haploid and diploid embryos [The differentiation between haploid and diploid seeds is largely performed visually, based on Purple Embryo Marker , which i embryos .R1-nj is affected by multiple factors, such as environmental conditions and donor genetic background [R1-nj expression, such as c1 inhibitor (c1-I), c2 inhibitor diffuse (c2-Idf) and intensifier1 dominant (in1-D) [Expression of ckground ,13,14,15 (in1-D) ,17,18. H (in1-D) ,20.Pl1 gene, which leads to light-independent, anthocyanin production in seedlings roots, was introgressed into some inducers because it serves as an additional mechanism for haploid and diploid differentiation. When present along with B1, R1-r or r1-1, Pl-1 will also induce anthocyanin production in seedling coleoptiles, leaf tips, margins and sheaths [B1 and Pl-1 will also lead to a dark purple pigmentation on husks and culm [The  sheaths . Jointlyand culm . Adult hand culm . TherefoEnvironmental conditions, such as temperature and relative humidity, likely impact HIRs ,6,13,23.1 or F2 plants of the haploid inducer RWS/RWK-76. Mean HIRs ranged from 0 to 11.3% using tropical donors [The term inducibility is used to describe the impact that the donor parent has on HIRs . Differel donors . Due to the complex influence that both inducer and donor parents have on HIRs, it is possible that there is an interaction between inducer and donor genetic backgrounds affecting the HIR. Highly significant genotypic differences were detected among inducers and source germplasm for HIRs in tropical conditions, but no interactions were observed between the two factors . Howeverp < 0.001) on the HIR .All factors considered had a highly significant effect ( the HIR . There wBoth screeners (11.7) and residuals (8.2) explained the variance in the HIR more than any interactions between year, donor, and inducer .RWS- and Mo-17-derived inducer backgrounds had higher average induction rates than the other inducers studied . OverallThe commercial dent hybrid background showed a relatively higher inducibility (9.66) as compared to other donors . In contOur primary aim was to determine if inducers belonging to different genetic backgrounds perform better on specific donor backgrounds. From the data presented in The trend lines show that there were few changes in ranking for different backgrounds, with the exception of sweet corn, for most inducers. Overall, the RWS inducer was superior. The PHI inducer performed better in sweet corn background, but performed the worst for the commercial dent hybrid, stiff stalk and non-stiff stalk donors. The B73-derived inducers had the highest interaction with donors performing second in commercial dent hybrid, third in non-stiff stalk and stiff stalk, highest in the flint donor and lowest in sweet corn. However, inducers did not differ statistically for the two poor-performing donors (sweet corn and flint) .R1-nj phenotype. This system is highly dependent on many factors including inducer lines, the inducibility of donor background, and environmental conditions [DH technology consists of generating haploid seeds from crosses of inducer lines with donors of interest followed by selection of haploids based on the nditions . In our nditions ,6.R1-nj expression, leading to a high misclassification rate. Presence of dominant anthocyanin inhibitor genes such as C1-I, C2-Idf, and In1-D in donor backgrounds or dosage effects can make this marker ineffective in haploid selection [C1-I, which are common in flint maize, are present, R1-nj color marker expression is completely suppressed and haploid seed identification is almost impossible [R1-nj expression were observed in the majority of crosses between inducers and commercial sweet corn hybrids [1 or F2 populations are used as source materials and when only one parent has inhibitor genes, seeds will segregate for the Navajo phenotype. In such cases, one may not be able to identify all haploid seeds efficiently and could potentially lose half to three-quarters of the haploids [However, the flint and sweet corn backgrounds showed overall lower HIRs compared to dent genotypes. Selection of haploid seeds in such backgrounds is hampered due to variation in election . If domipossible . Large v hybrids . When F1haploids .1 seeds or plants are misclassified as haploids (type II error), i.e., false negatives, given that the null hypothesis (H0) assumes that the seeds are haploids. A type I error may occur due to the limited efficacy of the R1-nj color marker or due to insufficiently trained technical staff. In our case, screeners caused the most variation  is similar in dent and flint materials [It was suggested  that foraterials . Eder anaterials  found anThe interaction plot suggests that the RWS inducer performed better than other inducers in all donors considered in this study. In commercial dent background, most of the inducers bar PHI had a higher HIR than in other backgrounds. For non-stiff stalk backgrounds, RWS, Mo17-derived, B73-derived and A632.75/B15-derived inducers were equally efficient; for stiff stalk backgrounds, only RWS and Mo17-derived inducers showed better performance . The datThe sixteen inducers used in this experiment can be grouped into seven distinct backgrounds based on their pedigree. Twelve of the sixteen inducers were developed by the DH Facility of Iowa State University (DHF-ISU), and have inducers RWS and RWK-76 as their source of haploid induction ability. BHI305, BHI306 and BHI307 are near isogenic inducers developed from the inbreds A632.75 and B-15 dent sterile (A632.75/B-15-derived). BHI201, BHI101 and BHI103 are near isogenic inducers developed in from the inbred B73 (B73-derived). Three near isogenic inducers derived from the inbred Mo17 do not have commercial names, and are referred to here as Mo-15, Mo-17, and Mo-23 (Mo17-derived). Three near isogenic inducers derived from the inbred LH82 also have no commercial names, and are referred to as LH82-26, LH82-28 and LH82-29 (LH82-derived). The following inducers were developed in Europe. RWS was developed at the University of Hohenheim, by crossing inducers WS14 and KEMS [1 hybrids were created within the Lancaster  heterotic group by crossing the inbreds PHN82 with PHP76, and PHG29 with PHG83. Two F1 hybrids were created within the stiff stalk synthetic heterotic group, by crossing the inbreds PHG86 with PHW17, and LH206 with PHW52. Two F1 flint hybrids, LFN1971.LGR2038 and LFR1941.19944, belonging to MAS Seeds, were used to represent the flint group. The reason for using hybrids rather than inbreds was due to their higher seed set, which has an impact on the power of the statistical analyses.The eight donors used in this experiment can be divided into five different genetic groups. Viking 60-01N is a commercial dent corn hybrid developed by Albert Lea Seed and is presumably derived from a cross of two inbred lines belonging to different heterotic groups. Golden Jubilee is a commercial sweet corn hybrid also developed by Albert Lea Seed . Two FThis experiment was conducted during the summers of 2016, 2017 and 2018 at the Iowa State University Agricultural Engineering and Agronomy Farm, located in Boone, Iowa. To ensure nicking, during the summers of 2016 and 2017, inducers were planted on a single planting date, while donors were planted on two distinct planting dates. Pollination was preferably performed on donor plants that presented fresh silks, which were trimmed to a size of 2 cm before pollination. During the summer of 2018, inducers and donors were planted on two planting dates, with the earliest planting of inducers used to pollinate the earliest planting of donors, and the latest planting of inducers for the latest planting of donors. During the summers of 2016 and 2017, inducers and donors were planted in 3.8 m-long plots. Each inducer had one planting with four rows and each donor had two plantings with two rows with twenty seeds per row. During the summer of 2018, each inducer and donor was planted in one 5.4 m-long plot, on two planting dates, with twenty-five seeds per row. Row spacing was of 0.76 m during all years. All trials were grown on loam soils, under rainfed conditions and adopting standard agronomic practices for maize production in Iowa. Pre-emergent herbicides and hoeing were used for weed control.All donor plants were manually detasseled and shoots were covered using glassine bags before silk exposure. Bulk pollen of each inducer was collected in brown tassel bags and used to pollinate at least ten ears of each donor genotype. Each donor plot was properly labeled and pollinated by a single inducer. All pollinated ears from a plot were bulk harvested when seeds reached the harvest maturity stage.R1-nj marker [Pl-1 allele, which leads to anthocyanin production on seedling roots. Putative haploids generated in crosses with these inducers were planted in the greenhouse and verification was performed using the root color marker. For all other inducers, putative haploids were planted in the field and their ploidy verified using the gold-standard test based on visual assessment of differences in plant vigor, erectness of leaves, and male fertility in haploids and diploids [The ploidy level of the embryo of each seed was evaluated using the j marker . Each eadiploids . These pThe HIR of each ear was corrected by multiplying the calculated HIR with the frequency of true haploid plants observed using the red root marker or based on the gold-standard test ,22. Dataijklmn is the HIR on the ith year, by the jth screener, from the mth inducer from the kth inducer background and the nth donor from the lth donor background; μ is an overall effect, bi is the fixed block  effect, sj is the random screener effect, αk is the inducer background main effect, βl is the donor background main effect, and (αβ)kl is the interaction effect. gikm is the random inducer effect (nested within inducer background), g′iln is the random donor effect (nested within the donor background), g″iklmn is the random interaction effect between the donor and the inducer (nested within their respective backgrounds), and ϵijklmn is the residual error.The statistical model used for the analysis is described below:p-value plots, and pairwise p-value matrices. Statistical analyses and visualization were conducted using R [Following model fit, the marginal means of inducer backgrounds, donor backgrounds, and their combinations were obtained and Tukey’s adjusted pairwise comparisons were performed. Pairwise comparisons were summarized by connected letters reports, pairwise  using R .p < 0.001) on the HIR. RWS and Mo-17-derived inducer backgrounds had higher average induction rates than the other inducers studied. The commercial dent hybrid was determined as a donor with high inducibility. Sweet corn and flint corn showed low inducibility, with no statistical difference between the inducers. Non-stiff stalk and stiff stalk backgrounds showed intermediate inducibility. More extensive studies with more donors and germplasm groups are needed to identify the best-matched inducers. Anthocyanin inhibitor genes in poor-performing donors were assumed to have increased the misclassification rate in the F1 fraction, which led to a lower HIR.Inducer and donor backgrounds considered in this experiment had a significant effect (