Corneal confocal microscopy (CCM) is a rapid non-invasive in vivo ophthalmic imaging technique that images the cornea. Historically, it was utilised in the diagnosis and clinical management of corneal epithelial and stromal disorders. However, over the past 20 years, CCM has been increasingly used to image sub-basal small nerve fibres in a variety of peripheral neuropathies and central neurodegenerative diseases. CCM has been used to identify subclinical nerve damage and to predict the development of diabetic peripheral neuropathy (DPN). The complex structure of the corneal sub-basal nerve plexus can be readily analysed through nerve segmentation with manual or automated quantification of parameters such as corneal nerve fibre length (CNFL), nerve fibre density (CNFD), and nerve branch density (CNBD). Large quantities of 2D corneal nerve images lend themselves to the application of artificial intelligence (AI)-based deep learning algorithms (DLA). Indeed, DLA have demonstrated performance comparable to manual but superior to automated quantification of corneal nerve morphology. Recently, our end-to-end classification with a 3 class AI model demonstrated high sensitivity and specificity in differentiating healthy volunteers from people with and without peripheral neuropathy. We believe there is significant scope and need to apply AI to help differentiate between peripheral neuropathies and also central neurodegenerative disorders. AI has significant potential to enhance the diagnostic and prognostic utility of CCM in the management of both peripheral and central neurodegenerative diseases. Peripheral neuropathies are highly prevalent neurological disorders which result in motor, sensory and autonomic disturbances, but can pose significant diagnostic challenges . Their eThe diagnosis of peripheral neuropathies has relied heavily on identifying neuropathic symptoms and neurological deficits on neurological examination . Questio2 of the corneal surface [The human cornea is the most densely innervated tissue in the human body, receiving sensory innervation from the ophthalmic branch (V1) of the trigeminal ganglion. The trigeminal ganglion contains approximately 27,400 pseudo-unipolar neurons of which ~1.3% innervate the cornea . Nerve b surface . These C surface . Early r surface .CCM is a rapid, non-invasive, and reiterative technique to image the cornea at 600× magnification in the clinic, without the need for expensive or time-consuming laboratory analysis. CCM has been extensively used in ophthalmology practice to diagnose and manage corneal disease and to monitor wound healing following surgery. Over the past 20 years, our group has pioneered the use of CCM as a biomarker of peripheral neuropathy. In 2003, we demonstrated that CCM identifies subclinical and progressive loss of corneal sub-basal nerves with increasing severity of diabetic neuropathy . Subsequn = 220) using a higher optimal threshold than the consortia paper (CNFL < 15.3 mm/mm2) reported 0.8/0.59 sensitivity/specificity for diagnosing DPN [CCM has a growing body of evidence demonstrating that corneal nerve fibre loss is a validated surrogate biomarker of peripheral neuropathies and central neurodegenerative diseases ,30. CCM sing DPN . CNFL prsing DPN ,26, thussing DPN  recentlysing DPN . Our datsing DPN , especiasing DPN ,45.CCM has been used to identify corneal nerve loss in a range of peripheral neuropathies including idiopathic small fibre neuropathy (ISFN), CIPN, chronic inflammatory demyelinating polyneuropathy (CIDP), HIV-associated neuropathy, hereditary sensory-motor neuropathies ,51,52,53Corneal nerve pathology has been demonstrated in patients with Parkinson’s disease, multiple sclerosis (MS), dementia ,65,66 anNon-overlapping (<20%) images are acquired from the corneal apex  and 5–8 images are selected based on the depth and quality to ensure accurate and representative analysis . QuantitManual analysis relies on expert manual annotation and tracing of corneal nerves to delineate main nerve fibres, branch points and fibres. Dabbah et al.  developeAutomated CCM image analysis software evaluates nerve fibres through segmentation and quantification of corneal nerve morphological features. Dabbah et al.  developeComplex geometric structural properties can be characterised by different scale values, namely fractal geometry . A fractThe architecture of the retinal microvasculature has been quantified using fractal analysis in patients with diabetes ,96,97. CCorneal nerve geometry can be quantified using corneal nerve fractal dimension (CNFrD) . CCM imaThe complexity of corneal nerve architecture is altered in DPN . The diaArtificial intelligence (AI) is a major field of study that aims to develop computers and machines that can emulate aspects of human intelligence. Machine learning, a sub-field of AI, describes a large and diverse set of algorithms that learn through examples and data. Artificial neural networks (ANN) are machine learning algorithms structured much like biological neurons. ANNs can be multi-layered to improve their performance on complex tasks which is termed deep learning. The non-invasive nature of CCM, and the ability to capture a large quantity of images leaves it well positioned for use in the AI revolution for data-driven disease modelling.AI aims to automate complex tasks normally performed by humans and is best understood as an umbrella term, with DLA being a specific type of AI. DLA are multi-layered artificial neural networks (ANN) whose architecture is inspired by biological neurons . Each ANOphthalmology lends itself well to AI due to the diverse range of digital imaging modalities and large datasets produced during standard clinical care. DR, age related macular degeneration (ARMD), retinopathy of prematurity (ROP), glaucoma and cataracts have all been identified as promising disease areas for AI . A systeWith the increasing worldwide prevalence of diabetes and DR , solutioThe success of AI is underpinned by the availability of big data, high-performance computing resources such as graphics processing units (GPU), and open-source development libraries . DLA, such as CNNs, have demonstrated comparable, and at times superior performance compared to human experts in a plethora of applications. Hence, DLA have been developed and validated to analyse CCM images.The non-uniform illumination or imbalanced intensity in CCM images poses challenges in evaluation for the diagnosis of diseases. Ma et al.  investigStandard CNN models  will typFor CCM image acquisition, each patient has multiple images taken, and depending on patient factors, the number of images per patient may vary. Some images contain discriminative and trustworthy information that can be used for classification of disease, while others do not. Therefore, it is critical to automatically select the trustworthy and discriminative images which will contribute to the model’s final prediction.CA-MIL (Consensus-Assistant Multiple Instance Learning) can take an arbitrary number of CCM images per patient as it’s input. This can strengthen the upper-bound ability of the DLA model, as more input information is assessed during the feature learning and extraction process.There is a consensus-assistant module that verifies the reliability of selected CCM images. Random noise (such as Gaussian noise) is inserted into the model along with the input CCM images to adversely perturb the model’s feature learning and prediction process. In other words, the predictions of a generalisable MIL classifier should be robust enough not to be influenced by input perturbations, and if the predicted class score changes significantly under a certain perturbation, this suggests an unreliable CCM image .This differs from classic MIL-based methods  that treConsidering the mass adoption and regulatory perspective, the ‘black-box’ nature of AI has been subject to resistance by some in the medical community . By the CCM identifies small nerve fibre damage in early DPN ,136 befoAI has the potential to fully unleash the full ability of CCM for the diagnosis of DPN, peripheral neuropathies and other neurodegenerative diseases. The incorporation of novel corneal nerve parameters including CNFrD, and the assessment of nerve complexity may provide unique and distinct disease-specific topographical signatures. AI-driven pathways for the identification of neurodegeneration will facilitate earlier diagnosis and improved monitoring of the effectiveness of interventions.


The cornerstones of sustainable development require the treatment of wastes or contaminated streams allowing the separation and recycling of useful substances by a more rational use of energy sources. Separation technologies play a prominent role, especially when conducted by inherently environmentally friendly systems such as membrane operations. However, high-performance materials are more and more needed to improve the separative performance of polymeric materials nanocomposites are ideally suited to develop advanced membranes by combining organic polymers with suitable fillers having superior properties. In this area, polyether block amide copolymers (Pebax) are increasingly adopted as host matrices due to their distinctive properties in terms of being lightweight and easy to process, having good resistance to most chemicals, flexibility and high strength. In this light, the present review seeks to provide a comprehensive examination of the progress in the development of Pebax-based nanocomposite films for their application in several sensitive fields, that are challenging and at the same time attractive, including olefin/paraffin separation, pervaporation, water treatment, flexible films for electronics, electromagnetic shielding, antimicrobial surfaces, wound dressing and self-venting packaging. It covers a wide range of materials used as fillers and analyzes the properties of the derived nanocomposites and their performance. The general principles from the choice of the material to the approaches for the heterogeneous phase compatibilization as well as for the performance improvement were also surveyed. From a detailed analysis of the current studies, the most effective strategies to overcome some intrinsic limitations of these nanocomposites are highlighted, providing guidelines for the correlated research. Nanocomposites are more and more present in our everyday life such as in the automotive sector, construction, anti-corrosion barrier coatings  and evenOriginally, inorganic–organic nanocomposites were developed to take advantage of the lightweight, flexibility and mouldability of a polymer matrix combined with the superior strength, chemical resistance and thermal stability of inorganic additives. However, the mutual interactions between the matrix and the additives result in performance overcoming those described by simple models based on only pure material properties.Inorganic–organic hybrid membranes, referred to as mixed matrix membranes (MMMs), represent a peculiar category of nanocomposites and are actively investigated for virtually all membrane operations. Indeed, MMMs effectively combine the properties of different materials, allowing to take advantage of even innovative materials  in molecular separations .®.Polyether block amides (PEBAs) are plasticizer-free thermoplastic elastomers (TPEs) . Their m® have been extensively investigated for membrane separation ) added to Pebax®5513 , the silver ions were not stabilised since the rigid amide block in the polymer matrix impeded the IL penetration, resulting in a poor propylene/propane separation performance compared to PEO polymers [SCN], [C2mim][BF4], [C2mim][OTf], [C2mim][Tf2N]) were prepared with an IL concentration from 20 to 60 wt% [BF4] and [C2mim][SCN] to attain the best separation performance and mechanical stability vs. feed pressure, whereas the highest permeability was reached at a higher IL content (60 wt%). The selective recovery of hydrofluorocarbons (HFCs) and hydrofluoroolefins (HFO) from exhausted refrigerant mixtures is a particular olefin/paraffin separation. This separation is required to recover and reuse high-global warming potential refrigerant gases, avoiding their emissions. Membranes combining Pebax 1657 with some ionic liquids , at Pervaporation (PV) is recognized as a green operation to carry out the purification process in chemical industries ,71. VolaJ), thickness-normalized permeate flux (NJ), separation factor (α), enrichment factor (β) and thickness-normalized pervaporation separation index (PSI) are the main variables employed to evaluate the PV separation performance of the membranes:W is the permeate amount collected in the period t, A is the effective membrane area, l is the membrane thickness, x and y are the weight fraction for the most permeable species in the feed and permeate stream, respectively.The PV application requires dense films. The permeate flux  ) leaching   nanoparticles that remarkably enhanced both flux and separation factor, with respect to the pure PEBA-based membranes.Hollow fibre mixed matrix composite membranes were prepared by a facile dip-coating method using ceramic supports and applied for recovering ethanol from an aqueous solution via pervaporation . The MMM2N], IL1) and N-octyl-pyridinium bis[(trifluoromethyl)sulfonyl]imide , are both hydrophobic due to the presence of two trifluoromethyl (-CF3) groups in the anion. The modified fillers were loaded within a Pebax matrix, preparing membranes for butanol/water PV. The mesoporous structure of the molecular sieves was preserved after the IL modification, as shown by XRD, TEM and BET analyses, thus it facilitated butanol and water diffusion in the membrane, significantly improving the permeation flux. The IL-modified particles enhanced the membranes’ hydrophobicity, particularly in the case of the MOFs modified with IL2 which present a longer alkyl chain on its cation. Accordingly, the presence of the IL-modified MCM-41 promoted the preferential sorption of butanol, enhancing also the separation factor of the MMMs. The optimum loading was 5 wt% for both fillers, while their agglomeration was observed at 10 wt%. The MCM-41-IL2 provided membranes with slightly better performance than MCM-41-IL1, with a higher degree of swelling and affinity for butanol, showing also high stability within 100 h of operation. The PV performance was improved at a high feed temperature, due to the enhanced diffusion for the small species and membrane free volume . . 38]. −2∙h−1 and a satisfactory furfural/water separation factor of 50.2). The MMMs’ performances were stable during the long-term operation (200 h) in a simulated hydrolysate containing furfural/acetic acid/water.The microwave synthesis remarkably reduced the synthesis times required to produce the MOFs, passing from several days to minutes. Nevertheless, the described preparation method allows for controlling the particle size and morphology. Good interfacial compatibility was observed owing to the chelating effect between the aluminium nodes of MOFs and the ether groups of PEBA, resulting in enhanced thermal stability. The MOFs, being hydrophobic and porous, boosted the affinity for furfural and the PV performances of the MMMs. The optimal performance was obtained using the coin-like particles  due to homogeneous distribution and molecular sieving provided by MIL-53(Al). A high PV performance was evidenced at a loading of 15%  were loaded into Pebax coating PVDF supports to prepare composite MMMs for the PV removal of phenol from aqueous solutions . The wat2  or graph2 ), the cr2 . Organic fillers were considered for producing MMMs in order to obtain a good interfacial affinity between fillers and polymer.Dense membranes for the ethanol separation from water were prepared by incorporating organic fillers -N-(pyridine-2-yl)benzamide (denoted as F1) and 4-(dimethylamino)-N-(pyridine-2-yl)benzamide (denoted as F2)) . These f+@SNW-1, is a COF with amino groups and a high amount of silver ions (48 wt%). Good interfacial compatibility between filler and polymer was obtained due to the organic nature of SNW-1. The channels within the SNW-1 filler introduced extra free-volume cavities within the membrane, improving the diffusion rate of the penetrants. On the other hand, the facilitated transport sites in the membranes resulted in enhanced sorption selectivity due to the reversible π-complexation interaction between Ag+ and thiophene molecules [DCA]) were loaded into Pebaxr vapour . Indeed,®3533, polyacrylic acid (PAA) and cellulose nanocrystals (CNCs) in an electrospinning device [DMSIP] that present a long alkyl chain in the cation provided an interesting antimicrobial activity, combining hydrophilicity, permeability and thermal stability. Instead, the smaller IL2 is readily released from the films without altering the hydrophobic nature of the neat polymer. Pebax casting . Pebax®R2 day) to 2350 g/(m2 day) at 40% CPD [2/day) and permeability (WVP = 16.4 g μm/m2 day Pa) to be used as breathable films for environmental  or biomedical  applications [Sole et al. produced breathable membranes by blending Pebax MH 1657 with different organic molecules ,61,62. D 40% CPD . However 40% CPD . Instead® nanofibers containing Ag nanoparticles produced by electrospinning [3 and the obtained samples were treated with UV light to reduce the silver ions to metallic Ag nanoparticles. The Ag nanoparticles (15 nm in size) were uniformly distributed in the Pebax fibres and decreased the polymer crystallinity as well as the onset temperature for decomposition. A very low amount of Ag salt was used (0.05–0.25‰) by Liang et al. [3 in Pebax® (0.15‰) the inhibition rate was >99.9% and the antimicrobial activity against Escherichia coli and Staphylococcus aureus was 5.8 and 5.6, respectively [Antibacterial materials were obtained as Pebaxspinning . In partg et al. . No antiectively . This AgPebax-based nanocomposites were tested for the packaging sector. One of the requirements for this application is stability under electron beam irradiation since this is a common method to sterilise pharmaceutical packaging products and medical devices. Virgin Pebax, exposed to various electron beam and gamma irradiation doses, presented branching and chain scission . The rad® MH1657 with polyethylene glycol dimethyl ether (PEGDME) and/or hydroxyl-terminated polyethylene glycol (PEGOH) [® and PEG were totally miscible for a PEG amount less than 30 wt%. This study demonstrated that a wide range of films could be prepared with tailorable mechanical and gas transport properties, capable to prolong the shelf-life of highly breathable fresh products.Thin transparent and breathable films were prepared for food packaging applications by the solvent casting method combining Pebax (PEGOH) . PEG decSelf-venting packaging materials were obtained by adding halloysite nanotubes (HNTs) to a matrix composed of Pebax and polyethylene glycol (PEG) . The filDifferent studies investigated the behaviour of Pebax nanocomposite structures via a comprehensive characterization. 2) [2 processing leads to a reduced crystallization rate. The decreased crystallite size (from 4.49 nm for neat Pebax to 3.48 nm for PebaxSCF) indicates that scCO2 processing involves a rearrangement of polymer chains to kinetically more favourable configurations, improving homogeneity between amorphous and crystalline parts of the Pebax copolymer. At the same time, graphene exfoliation is promoted. Nanocomposites based on Pebax and graphene were examined by analysing their crystallization kinetics during the extrusion process with and without supercritical fluid carbon dioxide (scCO2) . The scCAmong the most recent trends, wood/polymer nanocomposites, materials for protection against chemical warfare agents (CWAs) and materials for microelectronic cooling were developed as detailed below.® copolymers that can interact with the wood fibres. The thermal stability of the composites displayed a noteworthy improvement under air atmosphere. Therefore, the loading of wood fibres in Pebax® delays the thermo-oxidation in air due to the formation of char residue in the earlier stages of degradation. Tensile strength, modulus and impact strength were significantly increased upon the addition of nanolignocellulose  to Pebax by compression moulding using a hot press [Sliwa et al.  studied ot press . These nPebax was successfully adopted to improve the dispersion of boron nitride nanosheets in an epoxy resin . Pebax f2 and HKUST-1) to an inert PP substrate in order to develop materials for personal protective equipment against toxic chemicals [2) while the protection of activated carbon fibres is ∼150 min. Interestingly, the use of Pebax as an adhesive stabilises HKUST-1 to moisture. The adhesive properties of Pebax were exploited to attach MOF particles , while specific material combinations result in significantly better separation performance. The majority of the revised studies on membranes are related to the pervaporation process, investigating the influence of operating conditions such as feed temperature and concentration on process performance. Depending on the Pebax copolymer grade, both hydrophilic and hydrophobic membranes can be produced. However, the most used Pebax grade is the hydrophobic 2533 grade owing to the high amount of flexible polyether block. The design of the membrane structure and the choice of materials are key factors for achieving optimal performance. Supported membranes are the ideal configuration for pervaporation or water treatment owing to the increased flow rates obtained by thinner separative layers. Supported hollow fibres are also produced for pervaporation applications. On the other hand, the rubbery Pebax matrix well accommodates advanced materials capable of providing better separation properties. Therefore, several fillers with distinctive features are successfully dispersed in Pebax matrices, including metal oxide nanoparticles, carbon-based materials , clays, mesoporous silica, zeolites, Halloysite nanotubes and MOFs. The effects of the fillers on the morphology and separation properties of the resulting membranes were systematically studied, revealing that the loading amount has to be optimized in nanocomposite development. Indeed, low amounts of particles are capable to disrupt the intrinsic arrangement of the polymer chains, thus enhancing the available free volume in the matrix. Instead, higher loadings can induce a partial polymer chain “rigidification”, but inevitably result in the formation of agglomerates that typically cause a delay in the material performance. However, detrimental phenomena at the interface of heterogeneous phases are reduced by the use of compatibilising agents, capable of facilitating the incorporation of the filler within the host matrix  or by fillers having organic moieties .Novel strategies to design high-performance nanocomposite membranes include the manipulation of the filler’s morphology, leading to 2D configurations  or the use of fillers with a hierarchical structure  or with multifunctional properties . Among the different fillers investigated, those having porous structures introduce a further degree of freedom in the separation due to the additional molecular sieve mechanism. The extensive investigation of the microstructure-performance relationships of the produced MMMs explains the fundamental role of the filler morphology. Selected additives are capable to improve membrane performance with respect to benchmark materials  and reduce the swelling phenomena in membrane samples, increasing separation properties and physicochemical stability over time. In this respect, fillers shaped as 2D nanosheets proved particularly effective in improving the permeation and separation capability of the neat copolymers. Thus, membranes incorporating mesoporous nanosheets demonstrate considerable potential for industrial-scale application in both dehydration and humidification. This holistic review considers also the nanocomposites developed for a wide range of sensitive applications including electrochemical shielding, sensing devices, packaging films, antimicrobial materials, breathing tissues and biomedical devices such as wound dressings. Nanocomposite films having a porous structure were developed by electrospinning and successfully applied to adsorption processes or sensing. Flexible self-supported structures can be obtained for those applications that do not involve molecular-level separations. Indeed, Pebax copolymers present a tailorable combination of flexibility and partial rigidity of the matrix due to semicrystalline rigid polyamide domains in a continuous soft region of polyether that can be exploited in order to produce unprecedented materials, constructing highly conductive nanodomains for batteries or obtaining the filler segregation only in one copolymer phase. The chemistry of the two blocks permits selective interactions with the additives, thus resulting in reduced leaching of liquid additives  as well as excellent compatibility with solid organic and inorganic fillers. This work aims to inspire and motivate the development of nanocomposite membranes and materials also in novel applications covering the fabrication of membranes for high-performance lithium-ion batteries, films for electromagnetic shielding and protection against chemical warfare agents and materials for microelectronic cooling. Alternative uses of Pebax are as filler coating agents for improving the exfoliation of inorganic fillers within an epoxy matrix and as glue to assist the deposition of fillers on inert supports.The excellent progress achieved in recent years enables Pebax-based nanocomposites to become a versatile platform, endowing accessible approaches for molecular separation and sensing as well as for the rapidly growing additive manufacturing industry.


Recent studies indicated that the prognosis of patients with gastrointestinal tumors is frequently influenced by its complications, notably myocardial injury. The main object is to investigate the occurrence and risk factors of myocardial injury in patients with gastrointestinal tumor.1126 patients who received gastrointestinal tumor related surgery from May 2018 to June 2020 in the Sixth Affiliated Hospital of Sun Yat-sen University were retrospectively collected and divided into the non-myocardial injury group and the myocardial injury group (high-sensitive cardiac troponin I (hs-cTnI) ≥ 0.028 ng/ml). The occurrence and risk factors of myocardial injury in patients with gastrointestinal tumor are analyzed. The influence of myocardial injury on the ICU detention time in gastrointestinal tumor patients is also studied.P-value < 0.05). In addition, patients in the myocardial injury group also presented with a higher revised cardiac risk index (Lee index), higher neutrophil granulocyte ratio, lower hemoglobin, and higher likelihood of impaired cardiac structure and function . There was a trend of statistical significance in the ICU detention time between the myocardial injury group and the non-myocardial injury group . In this study, there were 7 patients presented with clinical symptoms in the myocardial injury group . In the multivariate analysis, advanced age, increased Lee index score, increased neutrophil granulocyte ratio, decreased left ventricular ejection fraction (LVEF), increased interventricular septum were independent risk factors for myocardial injury.In total, 78 (6.93%) patients developed myocardial injuries. Compared with patients in the non-myocardial injury group, patients in the myocardial injury group have a higher prevalence of cardiovascular risk factors (including advanced age and higher smoking ratio), a higher prevalence of comorbidities , and a higher rate of premedication  (all with In conclusion, advanced age, increased Lee index, increased neutrophil granulocyte ratio, decreased left ventricular ejection fraction, and increased ventricular septum were independent risk factors for preoperative myocardial injury in patients with gastrointestinal tumors. The proportion of clinical symptoms in gastrointestinal tumor patients with myocardial injury was low, indicating the necessity to closely monitor the cardiac status of individuals with gastrointestinal tumors.The online version contains supplementary material available at 10.1186/s12872-023-03086-1. Gastrointestinal tumor has become one of the most prevalent tumors around the world, affecting millions of people worldwide , 2. EarlHs-cTnI (high-sensitive cardiac troponin I) is a index with high specificity and sensitivity for the detection of myocardial injury, which can reflect the myocardial injury of non-cardiogenic diseases. Elevated levels of hs-cTnI is associated with higher mortality in non-cardiac hospitalized patients –6, incluHowever, to the best of our knowledge, no studies have reported the incidence and risk factors of myocardial injury in patients with gastrointestinal tumors. This is a retrospective study to investigate the risk factors leading to myocardial injury in patients and thus screening out patients with high-risk gastrointestinal tumors complicated with myocardial injury.This was a single-center retrospective study. Patients who admitted to the Sixth Affiliated Hospital of Sun Yat-sen University from May 2018 to June 2020 and had been tested for hs-cTnI were screened. 1126 consecutive patients who compliance with inclusion criteria and were finally enrolled  who underwent tumor related surgery under general anesthesia and completed hs-cTnI testing at admission within 7 days before surgery were consecutively enrolled. Exclusion criteria were emergency surgery, failure to received tumor related surgery, and clinical evidence of acute coronary syndrome  in the medical history of preoperative assessment. The database of this study comes from the study published in EClinicalMedicine in August 2021 , and classification variables are expressed as frequency (percentage). Continuous variables were compared using the independent T-test or paired sample T test or Mann–Whitney U test, and categorical variables were compared using the Chi-square test or Fisher's exact test where appropriate. Odds ratios for candidate variables were calculated by univariate logistic regression analyses. The indexes with significant differences were analyzed by multivariate logistic regression, and a The final sample size consisted of 1126 patients . Compared with patients in the non-myocardial injury group, patients in the myocardial injury group have a higher prevalence of cardiovascular risk factors (including senior age and higher proportion of smoking), a higher prevalence of comorbidities  . As compared with non-myocardial injury group, patients in the myocardial injury group also presented with a higher revised cardiac risk index (Lee index), higher neutrophil granulocyte ratio, lower hemoglobin, and higher likelihood of impaired cardiac structure and function . In addition, 6.4% of patients from the myocardial injury group take statins, which is relatively higher than that in non-myocardial injury group. There was no statistical difference in the proportion of receiving preoperative chemotherapy between the two groups. , and there was a trend of statistical significance in the ICU detention time (1[P = 0.064) between the two groups.Moreover, there was a statistical significance in ICU admission between the myocardial injury group and the non-myocardial injury group .As shown in Table P = 0.005) and 2.378 , respectively.As shown in Table We found in the present study 78 cases (6.93%) of 1126 patients with gastrointestinal tumors were complicated with myocardial injury. Advanced age, increased Lee index score, elevated neutrophil granulocyte ratio, decreased LVEF, and increased ventricular septum thickening were identified as independent risk factors for gastrointestinal tumor complicated with myocardial injury.Previous studies reported myocardial injury occurs frequently 42/586, 7.17%) in patients undergoing colorectal tumor surgery in an enhanced recovery after surgery protocol [, 7.17% iOur study indicates that advanced age is a risk factor for preoperative myocardial injury in patients with gastrointestinal tumors. This is different from previous reports which showed age is not a risk factor for upper gastrointestinal bleeding with simultaneous myocardial injury . HoweverInflammation is recognized as a prominent feature of tumor progression . StudiesWe found that LVEF reduction and ventricular septum thickening were independent risk factors for gastrointestinal tumors complicated with myocardial injury. Previous studies have shown that hs-cTnI concentration is related to left ventricular diastolic dysfunction as suggested by echocardiography . TherefoWe found that there was a trend of statistical significance in the ICU detention time between the myocardial injury group and the non-myocardial injury group, which is consistent with the results of Tota-Maharaj et al.’ s study . The posThere are several limitations in this study. Firstly, this study is a retrospective single-center study, and the inclusion of gastrointestinal tumor patients undergoing surgical treatment does not represent the incidence of myocardial injury in all patients with gastrointestinal tumors. Prospective multicenter research is warranted in the future. However, our research suggests that gastrointestinal tumor is sometimes complicated with myocardial injury, which cannot be ignored in clinical practice. Secondly, there was no statistical significance between Lee index = 2 and Lee index = 0, which may be due to the small sample size of gastrointestinal tumor patients with Lee index ≥ 2. Thirdly, in order to better assess the cardiac status of gastrointestinal tumor patients and take necessary intervention before surgery, our present study focuses on the risk factors for preoperative myocardial injury. Therefore, risk factors for postoperative myocardial injury were not investigated in the present study.In conclusion, advanced age, increased Lee index, increased neutrophil granulocyte ratio, decreased left ventricular ejection fraction, and enlarged ventricular septum were independent risk factors for preoperative myocardial injury in patients with gastrointestinal tumors. The proportion of clinical symptoms in gastrointestinal tumor patients with myocardial injury was low, indicating the necessity to closely monitor the cardiac status of individuals with gastrointestinal tumors in preoperative period.Additional file 1. Chemotherapy information that shows the chemotherapy regimen and proportion of patients in each group.


Copsychus) that is assumed to be of least conservation concern. We uncovered cryptic diversity and an unexpected degree of hidden extinction and terminal endangerment. Our analyses reveal that >40% of the phylogenetic diversity of this radiation is already either extinct in the wild or nearly so, including the two genomically most distinct members of this group (omissus and nigricauda), which have so far flown under the conservation radar as they have previously been considered subspecies. Comparing the genomes of modern samples with those from roughly a century ago, we also found a significant decrease in genetic diversity and a concomitant increase in homozygosity affecting various taxa, including small-island endemics that are extinct in the wild as well as subspecies that remain widespread across the continental scale. Our application of modern genomic approaches demonstrates elevated levels of allelic and taxonomic diversity loss in a songbird clade that has not been listed as globally threatened, highlighting the importance of ongoing reassessments of extinction incidence even across well-studied animal groups.Quantifying the magnitude of the global extinction crisis is important but remains challenging, as many extinction events pass unnoticed owing to our limited taxonomic knowledge of the world’s organisms. The increasing rarity of many taxa renders comprehensive sampling difficult, further compounding the problem. Vertebrate lineages such as birds, which are thought to be taxonomically well understood, are therefore used as indicator groups for mapping and quantifying global extinction. To test whether extinction patterns are adequately gauged in well-studied groups, we implemented ancient-DNA protocols and retrieved whole genomes from the historic DNA of museum specimens in a widely known songbird radiation of shamas (genus Key words: extinction, introgression, white-rumped shama, conservation. The rate of global biodiversity loss has precipitously increased over the last few decades . This loMany studies have tried to quantify biodiversity loss using species as a currency to measure extinction rates . While tCopsychus malabaricus complex constitute a songbird radiation that is assumed to be relatively well-inventoried in taxonomic terms, although species boundaries have been shifting somewhat among the 16 taxa widely recognized in this complex  specimens exhibited high levels of post mortem damage in subsequent filtering steps and were also removed .In total, we successfully retained 59% (13 out of 22) of historic samples for genomic analysis after post-sequencing quality filtering, including from all taxa except Combining our historic DNA material with modern samples, we harvested 2,142,418 homologous single-nucleotide polymorphisms (SNPs) across all genomes. We concatenated all SNPs into one larger sequence and constructed a maximum likelihood topology using RAxML , calculajavanus and omissus clustered closely with each other, suggesting treatment as one Javan clade  instead of melanurus, its claimed affinity  and the remainder of the complex (a), with other taxon clusters emerging as distinct at lower PCs (b). Conversely, admixture analysis using NGSadmix, which was carried out from K = 2 to K = 15 (omissus emerged as highly differentiated from the other taxa in terms of DXY and FST summary statistics (candd), although the picture was less clear for FST, presumably because the latter is sensitive to artifacts of differing sample sizes per taxon. The genomic nucleotide diversity of some insular taxa, especially omissus, leggei, and javanus, turned out to be only roughly half that of the nucleotide diversity of most other taxa (a).Principal component analysis (PCA) based on 2,579,997 SNPs was consistent with our phylogenetic analyses, yielding the deepest division between the Javan individuals , together with an individual from Cambodia (JF318) and two samples from Vietnam with unknown localities (NRM20036774 and NRM20036776). This clustering pattern is supported by the SNP-based tree, in which the aforementioned group of five individuals is sister to the remainder of macrourus samples  from Vietnam were consistently distinct from other The museum samples sourced from the Naturalis Biodiversity Center, Leiden, were often associated with brief or illegible locality information; while we can be certain that they are from the Barusan islands, we often do not know which island they were collected on  online. c oxidase subunit 1 (COI) mitochondrial divergence threshold of 2–3% has been postulated in the literature to provide corroboration for species-level recognition of closely related bird species (b: 3.80%) and the taxon nigricauda from Kangean Island  , leggei from Sri Lanka , and suavis from Sarawak  , the main continental clade , and the Barusan clade  dropped below 2% (A cytochrome- species . The dee: 3.29%)  online. : 2.96%)  online. below 2%  online.C. albiventris as a yardstick for mitochondrial divergence thresholds in this complex because it has consistently been classified as an independent species by the vast majority of historic and modern taxonomic treatises  diverged from the Andaman shama and fell outside the main continental clade of white-rumped shamas  in the complex, and the “evolutionarily distinct and globally endangered score” (EDGE) following t scores . The EDGmacrourus, suavis, melanurus, hypolizus) for which genome sequences from two time points approximately a century apart were available, to determine the change in diversity over the last 100 years. Many measures of genetic diversity are subject to sample size bias. In order to rule out potential biases, we measured nucleotide diversity and heterozygosity using representative male samples from each taxon and time point with two exceptions—the modern samples for macrourus and suavis. The sex of these two samples is likely male, but remains unknown and could potentially be female, even though the genetic diversity values of their Z chromosome do not suggest so (We measured nucleotide diversity and heterozygosity for four taxa (ggest so  online.hypolizus (fig. 3a and fig. 3a). Comparing these values with genome-wide heterozygosity values of other birds, the four shama taxa fare slightly better than other endangered species (fig. 3b and DXY and FST) between historic and modern samples were plotted across the genome, hypolizus displayed a particularly pronounced variance as compared with the other three taxa , with an increasing recognition of the importance of biodiversity conservation . On a prC. malabaricus complex have been limited to few markers whose signal was dominated by mitochondrial DNA (supplementary fig. S4aandbomissus and javanus) on the brink of extinction, which have never previously been considered to be a distinct species (nigricauda) now widely believed to be extinct in the wild. Our phylogeny also confirmed the distinctness of stricklandii from Sabah  as most basal within white-rumped shamas calls for immediate conservation action, as these two taxa had previously passed for weak subspecies and gone under the conservation radar. Meanwhile, the unique reproductive display and behavior of these eastern Javan shamas is well known in breeders’ and traders’ circles and is partly responsible for their great endangerment . A high ED of Javan members among Sundaic radiations is not entirely unique to Copsychus: examples of Javan taxa of high genetic or bioacoustic distinctness within their Southeast Asian species complexes include Mixornis babblers .There is also extensive gene flow between the three taxa within the “Continental” clade  online, Because of our extensive knowledge about their biology and taxonomy, birds are a widely-used model organism and indicator group in conservation. For instance, Endemic Bird Areas (EBAs) are used as general indicators of organismic endemism by conservation planners to focus efforts on areas with concentrations of unique species . Our genC. malabaricus complex are among the most widely kept and intensely traded birds in Asia. Despite their great endangerment and local extinction in certain regions, such as Indonesia and Malaysia, they have not been recognized as threatened by the IUCN Red List . Using genomic data and accounting for secondary gene flow across the complex archipelagic landscape of Southeast Asia, we show that the genomically most distinct unit within the complex—which has hitherto never been recognized at the species level—is close to extinction in the wild, and that 2–3 other deeply divergent lineages that can plausibly be recognized at the species level are already extinct in the wild , accompanied by a 2-fold increase in the percentage of ROHs across the genome . On the surface, this result would suggest a gain in population-genetic diversity in hypolizus, yet the present-day field situation points to a different scenario: hypolizus shamas probably went extinct in the wild around 2015 (hypolizus sample (SIM44) comes from a local trader from Simeulue who breeds multiple shama taxa. The inflated nucleotide diversity value is likely to be an artifact of cross-breeding and the infiltration of alien alleles from the mainland clade.Only zygosity a, accompund 2015 , and ourCompared with heterozygosity estimates of other birds sourced from the literature, the modern values of these four shamas are generally higher than those of threatened species elsewhere  online. The current conservation status of white-rumped shamas belies the results of our genomic analyses, which have uncovered cryptic diversity and paint a bleak picture of hidden extinction events and instances of terminal endangerment. Multiple clades within the white-rumped shama are being threatened by unsustainable wildlife trade and habitat degradation .nigricauda from the Kangean Islands, which emerged as the second most distinct taxon of the radiation (javanus and omissus) and the Barusan clade , the latter two of which are either extinct in the wild or nearly so , followed by one species-level lineage that is terminally endangered (omissus), as well as additional species-level lineages of concern .To facilitate urgent conservation action, we present a new taxonomic classification based on our genomic data supplemented by insights from morphology and reproductive behaviour see . Our newConservation in many countries, especially in Southeast Asia, is often constrained by taxonomic bias, affording attention only to species-level lineages. Imperfect or erroneous taxonomic classifications will often lead to endangered species being ignored or left out of conservation planning . It is hC. malabaricus complex following the taxonomy of mirabilis.We obtained a total of 22 historic toepad samples and 34 modern tissue samples from various museums and through fieldwork conducted in the Indonesian archipelago  online. The DNA of modern tissue samples was extracted using the DNeasy Blood and Tissue Kit  following the manufacturer’s protocol with an additional RNase treatment applied. Toepad samples from dried historic skin specimens were extracted for DNA under sterile conditions inside a dedicated ancient-DNA facility using the same kit with slight modifications as previously documented .Fresh modern DNA was sheared into a targeted size of 250 bp using a Bioruptor Pico sonication device . Whole-genome libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina  for both fresh and historic samples, with additional modifications for the latter following Multiple extraction and library negatives were included to detect contamination. DNA concentrations were ascertained using a Qubit 2.0 high sensitivity DNA Assay kit , and fragment sizes were assessed using an AATI Fragment Analyzer . The negatives were also quantified using a Qubit 2.0 assay and AATI to ensure absence of contaminating DNA. Once checked, libraries were sequenced at NovogeneAIT Genomics (Singapore) on the Illumina Hiseq 4000 platform to produce 150-bp paired-end reads.C. sechellarum assembly (GenBank accession: GCA_013398635.1) using BWA-MEM (http://broadinstitute.github.io/picard/) was subsequently used to assign read group information and mark duplicates. Lastly, we used RealignedTargetCreator and IndelRealigner as implemented in the Genome Analysis Toolkit (GATK) v3.8-0    to realite, USA)  for mappte, USA)  online.Aerodramus swiftlet genome (unpublished data) because swiftlets were sequenced on the same Illumina lane. Using Fastq_screen  human (GenBank Assembly Accession: GCA_000001405.28), (ii) Seychelles magpie-robin (GenBank accession: GCA_013398635.1), (iii) a compilation of all available bacterial genomes on the RefSeq database, and (iv) an q_screen , we extrpost mortem DNA damage that can confound downstream analysis, necessitating the application of sophisticated analytical tools  before tree search. We created a maximum likelihood tree with the resulting dataset comprising 2,142,418 concatenated SNPs using the ASC_GTRGAMMA model in raxmlHPC-PTHREADS v 8.2.12 for 100 bootstraps with an oriental magpie-robin C. saularis as an outgroup  . SNPs wioutgroup .omissus), (ii) Kangean Island (nigricauda), (iii) Sabah (stricklandii), (iv) remaining Borneo (suavis), (v) Sri Lanka (leggei), (vi) Barusan Islands , and (vii) mainland tropical Asia , with oriental magpie-robin as an outgroup  East Java , (ii) Kangean Island (nigricauda), (iii) Sabah (stricklandii), (iv) remaining Borneo (suavis), and (v) mainland tropical Asia  with the oriental magpie-robin as an outgroup. These samples were remapped to the Collared Flycatcher Ficedula albicollis genome for chromosomal information (RefSeq accession: GCF_000247815.1). We then called SNPs again only for the major chromosomes before removing linked SNPs with a pairwise linkage disequilibrium correlation coefficient higher than 0.1. The resulting 463,621 SNPs were inputted into qpBrute for fitting admixture graphs. Bayes factors for each admixture graph were then calculated with a burnin of 1,800,000 and 9,000,000 iterations. Convergence was determined using the Gelman-Rubin convergence diagnostic and likelihoods calculated. A difference in likelihood values <3 was used as a benchmark for rejecting models.The maximum likelihood tree suggested the presence of incomplete lineage sorting or introgression in certain taxa (see Results), prompting us to investigate secondary gene flow using qpBrute . We reduK = 15  were calculated using pixy . We assessed population division in the radiation by running PCA in PCAngsd  and admiK = 15 . Populating pixy .C. saularis (GenBank Accession: NC_030603) using BWA–MEM during initial filtering. We employed ANGSD to call consensus sequences for each sample using -doFasta 3 (https://www.ncbi.nlm.nih.gov/orffinder/) to ensure the same orientation across all sequences and the absence of nuclear genes that may have been mistakenly included in the extraction. Following a quality check, we managed to retain all taxa including barbouri and albiventris.We also harvested the mitogenome from our samples by mapping the reads to the complete mitogenome of oFasta 3 . These sMAFFT was used to align the mitochondrial sequences, and samples with more than 30% missingness were removed . We calcC. malabaricus and Andaman shama C. albiventris). Therefore we assigned conservation status assessments to each of the lineages ourselves, using the IUCN status criteria and the best field knowledge of the day. For example, within the Barusan clade , most populations are now considered extinct in the wild, with the exception of melanurus on Siberut, which was still found in single digits in 2018 . Only a single individual was heard during the last expedition, possibly the last remaining individual on this small island , Bayesian approaches proved too computationally intensive and time consuming to run. We instead opted to use a non-Bayesian dating method, RelTime-OLS, using a relaxed clock as implemented in MEGAX that is known to be fast and accurate . We then in 2018 . The Easnd trade . As suchl island . Therefomacrourus, suavis, melanurus, and hypolizus—we tracked population-genetic parameters over the course of a century. In each of these taxa and time cohorts, the sample with the best DNA sequence quality was chosen if multiple individuals were available, with male samples being prioritized. The coverage and missingness across each sample are comparable to rule out potential biases.In the four taxa that were represented by both historic and modern samples—https://github.com/simonhmartin/genomics_general) to calculate nucleotide diversity, FST and DXY of the historic population and modern population using a window size of 50 kb and a step size of 10 kb. Furthermore, we calculated heterozygosity values for each sample using ANGSD based on a folded site frequency spectrum estimation.We conducted the following analyses 1-on-1 per taxon to minimize the effect of differing population sizes on our results. We carried out variant calling using ANGSD  for each taxon. We used the script popgenWindows.py . We generated genomic vcf files using bcftools and the above-mentioned reference genome. PLINK was used to calculate ROHs for each taxon and time cohort following the recommended filters (–homozyg-density 25 –homozyg-gap 500 –homozyg-het 15 –homozyg-window-snp 50 –homozyg-window-het 15 –homozyg-window-missing 1 –homozyg-window-threshold 0.05 –homozyg-snp 50 –homozyg-kb 300)  .msac189_Supplementary_DataClick here for additional data file.


Future treatment trials should consider stratifying patients using a combination of these high-risk biomarkers. In the absence of trials, risk stratifying patients based on the presence of these markers may lend to more individualized clinical decisions. We aimed to summarize the studies that have investigated the relationship between biomarkers and their role in predicting recurrent stroke risk in intracranial atherosclerotic disease.Recurrent stroke risk secondary to intracranial atherosclerotic disease remains high despite aggressive medical treatment. This risk is further amplified in subgroups possessing biomarkers of hemodynamic insufficiency and potential for embolization, which have been shown to be independently and synergistically predictive of recurrent stroke. Luminal stenosis was predominantly used as entry criteria in major treatment trials, discounting the potential role of hemodynamics from primary analyses, limiting the strength of evidence and conclusions of these biomarkers to  Intracranial atherosclerotic disease (ICAD) is the most common cause of ischemic stroke globally with the highest prevalence in Asians, Africans, and Hispanics , 2. The Although these studies targeted higher risk patients by including patients with at least moderate-to-high-grade stenosis, there is likely a high degree of heterogeneity within this group. For example, patients with a dominant hemodynamic mechanism may be at a greater risk of recurrent stroke compared to patients with thromboembolic mechanisms –12. SeveWe searched the terms: “Intracranial atherosclerotic disease”; “ICAS”; ICAD”; “hemodynamics in intracranial atherosclerotic disease”; “risk stratification of intracranial atherosclerotic disease”; WASID; SAMMPRIS; MyRIAD; TOSS-2; “embolic signaling in intracranial atherosclerotic disease”; “hypoperfusion in intracranial atherosclerotic disease” in Pubmed.Ischemic events that localize to a downstream atherosclerotic intracranial large artery narrowed to at least 50% of its vessel diameter can be classified under the stroke mechanism of ICAD . Althougpost-hoc analyses of the major trials below:Ischemic strokes attributable to ICAD can occur due to various mechanisms, including hemodynamic ischemia, artery-to-artery embolism, branch atheromatous disease, and a combination of these mechanisms. There is likely a complementary relationship between these mechanisms, since hypoperfusion likely increases thrombosis and decrease the distal washout of thromboemboli –24. Infapost-hoc analysis which found a strong relationship between the degree of stenosis and angiographic collaterals, and an inverse relationship between angiographic collaterals and anterograde flow across a stenosis (TIMI score) and into the downstream territory (TICI score)  . This costenosis . Althougstenosis . Artery-post-hoc analysis of anterior circulation infarctions, patients with qualifying events attributable to internal and cortical borderzones were at significantly higher rates of recurrent infarction compared to non-borderzone infarctions at a median follow-up of 31 months  (p = 0.014) and were found in 70% of patients with borderzone infarctions. The presence of a borderzone pattern coupled with impaired collaterals had the highest rate of recurrent infarction at 37%. In addition, the rate of recurrent infarction continued to increase beyond 1 year in patients with either borderzone patterns or impaired collaterals while rates remained steady in patients with non-borderzone patterns or complete collaterals. Among patients with borderzone infarctions as the qualifying event, the primary endpoint was lower in the stenting (18%) vs. medically managed group (26%), and a Kaplan-Maier curve of primary endpoints using this subgroup favored endovascular treatment, although this was not statistically significant . This study confirmed the strong relationship between angiographic collaterals and recurrent stroke as previously seen in the WASID post-hoc analyses above  . Impairelaterals .p = 0.04) with a hazard ratio of 11.55  in a risk factor-adjusted multivariate analysis which was resistant to the effects of disease severity and location. A related study was performed by the study group that found a correlation between vessel-specific flow and the severity of stenosis, however, distal flow status, incorporating collateral capacity, was not predicted by the severity or location of disease  study was the first prospective study to evaluate the relationship between hemodynamics and recurrent stroke risk in patients with ICAD . Vertebr disease . The VERp = 0.07). A post-hoc analysis showed that the baseline number of diffusion-weighted imaging lesions  and borderzone infarction patterns were significantly associated with new or recurrent infarction , implying that hypoperfusion and artery-to-artery embolism likely contribute to early subclinical infarction  and clinical recurrent stroke. Severe stenosis was associated with subcorti-cocortical pattern and multiple lesions on baseline imaging (p <0.001) but was not predictive of recurrent infarction.The Trial of Cilostazol in Symptomatic Intracranial Arterial Stenosis (TOSS-2) failed to show a difference in recurrent stroke between patients treated with dual-antiplatelet therapy vs. monotherapy . A subanSeveral retrospective studies have associated perfusion parameters and recurrent stroke risk in patients with intracranial atherosclerotic disease; Tmax >6s delays with mismatch volumes of at least 15 mL in the territory of a symptomatic intracranial artery were shown to be associated with recurrent cerebrovascular events , 18 and High resolution vessel wall imaging by MRI is a novel non-invasive imaging tool that has been used to distinguish ICAD from alternate stroke etiologies and to stratify stroke risk , 34. OurMicroembolic signals (MES) detected by transcranial doppler were first used as markers of plaque instability in extracranial symptomatic and asymptomatic carotid stenosis as MES correlated with several biomarkers of plaque morphology, including plaque ulceration  and neovSubclinical infarctions can occur due various stroke mechanisms  and haveIschemic strokes due to ICAD recur more frequently in the early period after the index stroke , 62, witRecurrent stroke risk in intracranial atherosclerotic disease remains high despite aggressive medical and endovascular therapies. Defining a vastly heterogenous disease exclusively based on luminal size discounts the role of other high-risk biomarkers, such as hemodynamics and potential for embolization, limiting the generalizability of current treatment trials. Incorporating these biomarkers in isolation as entry criteria into future trials may pose a challenge given the uncertain thresholds of hemodynamic insufficiency and thromboembolic potential. Perhaps the integration of biomarkers, given the probable synergism, may better target a hemodynamically insufficient subgroup most resistant to antithrombotic therapies, justifying endovascular flow augmentation, despite its current risks. While randomized controlled trials should remain the gold standard in guiding treatment, biomarkers of stroke recurrence may perhaps be used as an adjunct in clinical decision-making to better estimate recurrent stroke risk, allowing for more targeted and individualized treatment. The myriad of stroke mechanisms in intracranial atherosclerotic disease may be too complex and multidimensional to be managed by simplified and universal treatment strategies.AB and DL contributed to conceptualization, literature search, writing—original draft, and writing—review and editing. Both 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.


A common approach to phylogenomics is to extract related genes from each organism, build a multiple sequence alignment and then reconstruct evolution relations through a phylogenetic tree. Often a set of highly conserved genes occurring in single-copy, called core genes, are used for this analysis, as they allow efficient automation within a taxonomic clade. Here we introduce the Universal Fungal Core Genes (UFCG) database and pipeline for genome-wide phylogenetic analysis of fungi. The UFCG database consists of 61 curated fungal marker genes, including a novel set of 41 computationally derived core genes and 20 canonical genes derived from literature, as well as marker gene sequences extracted from publicly available fungal genomes. Furthermore, we provide an easy-to-use, fully automated and open-source pipeline for marker gene extraction, training and phylogenetic tree reconstruction. The UFCG pipeline can identify marker genes from genomic, proteomic and transcriptomic data, while producing phylogenies consistent with those previously reported, and is publicly available together with the UFCG database at  Fungi is one of the most diverse clades in the tree of life, potentially encompassing 2.2–3.8 million species  region of the nuclear ribosomal RNA (rRNA) cistron has long been used in phylogenetic analysis of fungi as the universal fungal marker  and has With the rapid increase of available public genomic sequences analysis using multiple markers became feasible, which increased the resolution further . The mosThere have been numerous efforts towards defining sets of single-copy orthologs for the fungal kingdom, notably the Fungal Genome Mapping Project , and thHere, we introduce the Universal Fungal Core Genes (UFCG), a database of fungal marker genes derived from experimentally annotated genes Figure  as well sp.).We obtained 12 027 whole genome assemblies of fungal species from the National Center of Biotechnology Information (NCBI) GenBank database  Figure . Taxonomab initio gene prediction using AUGUSTUS v3.4.0  against the full UniProtKB release 2022_03 , the maIn some cases, hits to certain genes may be fragmented into multiple smaller hits (e.g. due to the intron-exon structure of eukaryotic genes), causing them to be filtered out in downstream analyses. In order to recover such genes, we implemented a procedure in which we merge hits to the same gene occurring sequentially on the same genomic contig. If the distance between the start position of the first hit and the end position of the final hit gives ≥80% query coverage, the merge was considered valid.Genes that were identified as single-copy  from ≥95% of the 1587 species-representative assemblies were defined as candidate core genes, resulting in 62 genes.AUGUSTUS-PPX  providesIn each iteration, amino acid sequences of each gene are extracted from the species-representative assemblies with AUGUSTUS-PPX using the block profile HMMs from the previous iteration. Each extracted protein sequence was searched against the sequences from the respective MSA using MMseqs2. We accepted the protein sequence if its alignment covers at least 80% sequence length of a MSA member sequence. After each iteration a new MSA is generated by combining the previous and newly detected sequences using MAFFT v7.310 . The MSAFor the first iteration, we used block profile HMMs built from the query centered MSAs (described above) for prediction and the amino acid sequences from Swiss-Prot with the corresponding gene annotation for homology search validation. We conducted three iterations of AUGUSTUS-PPX training for each of the core gene candidates we defined earlier, resulting in a final set of block profile HMMs.To quantify the coverage of the core gene candidates on fungal species, we examined the presence of the genes from AUGUSTUS-PPX search against the species-representative genome assemblies. We repeated the final iteration of the profile HMM generation process described above to obtain the set of homologous protein sequences. The sequences with their alignment covering at least 80% sequence length of a member sequence of the respective MSA were accepted. A gene was defined present for the assemblies from which an accepted sequence was extracted.E-value lower than 10−3.For enhanced sensitivity, we relaxed the threshold and accepted the sequences covering at least 50% sequence length of a member. For the remaining sequences, the threshold was relaxed once more by accepting those aligned with We then tallied the proportion of the assemblies that reported the gene existing as a single-copy (i.e. only one homologous sequence detected), and those regardless of the copy number. Genes that ultimately failed to cover 95% of the species as a single-copy were rejected, while the remaining genes were defined as the final set of core marker genes.To benchmark the core genes, we used the same method to quantify the existence coverage against 9397 species-redundant genome assemblies, which passed the quality assessment but were unused because of their taxonomic redundancy.We found the absence of the conventional marker genes for multi-gene phylogeny from our computational investigation, due to their functional divergence and lack of universality across the entire kingdom ,39. To sWe developed a bioinformatics pipeline integrating the process of marker gene extraction and phylogenetic analysis in a fully automated fashion. The modular pipeline allows users to process their biological sequences into sets of marker genes, align marker gene sequences, concatenate gene alignments, construct phylogenetic trees, and train their own marker MSAs and profile HMMs Figure .profile, tree and train. The profile module accepts genome, transcriptome and proteome data as input, and extracts marker gene sequences with AUGUSTUS-PPX using pre-trained block profile HMMs. In addition to the UFCG markers, we prepared profile HMMs for the ITS region and 758 single-copy orthologs from the fungal subset of OrthoDB v10  as described above. A JSON file with valid amino acid and nucleotide sequences is produced as a result.We developed a pipeline with three main modules: hoDB v10  availabltree module gathers the collection of JSON files produced with the profile module, constructs MSAs for each shared marker gene using MAFFT  by fungal communities, resulting in 20 canonical genes , UniProse .data, genome_info, run_info, respectively), which are downloadable from the database. We organized them into a sortable and searchable table, which provides the download links along with their NCBI accession numbers and taxonomic annotations. Additionally, we extracted UFCG markers, ITS region, and BUSCO sequences from 9397 species-redundant genome assemblies, which are also downloadable from the database as compressed archives.The UFCG species database contains pre-extracted sequences of UFCG markers, ITS region, and BUSCO identified in the set of representative genome assemblies from 1587 fungal species as described Figure , bottom.The UFCG database will continue to be updated with enriched sequences and MSAs of marker genes and pre-extracted sequences as new NCBI GenBank releases become available.The UFCG pipeline can extract marker genes from assorted types of biological sequences, including DNA, RNA and protein. To illustrate this, we constructed a phylogenetic tree of UFCG marker genes extracted from 13 genomic, 8 transcriptomic and 13 proteomic sequences, originating from three species under the order Eurotiales , the placement of these pairs have been contentious throughout previous studies , however, which we speculate is due to the highly-conserved nature of the marker genes. In such cases, markers with higher resolution  can be easily trained into prediction models with the t, P = 9.57 × 10−10), implying the congruence of UFCG tree with BUSCO tree was predominantly derived from the core gene markers we defined rather than those canonically accepted.Additionally, we constructed separate phylogenies from the 41 core gene markers and the 20 canonical markers and performed the same comparison. Trees built from the core gene markers were significantly more congruent with the BUSCO tree compared to those built with just the canonical genes , AutoMLhttps://ufcg.steineggerlab.com. Entire content of the database is licensed under CC BY-SA 4.0. The pipeline is implemented in Java and is available as GPLv3 licensed free open-source software at https://github.com/steineggerlab/ufcg.The UFCG database is freely available without registration at gkac894_Supplemental_FileClick here for additional data file.


Most pregnant women in low and lower-middle-income countries do not receive all components of antenatal care (ANC), including counselling on obstetric danger signs. Facility-level ANC guidelines and provider in-service training are major factors influencing ANC counselling. In Ethiopia, little is known about the extent to which guidelines and provider in-service training can increase the quality of ANC counselling.We examined the effect of national ANC guidelines and ANC provider in-service training on obstetric danger sign counselling for pregnant women receiving ANC using the 2014 Ethiopian service provision assessment plus (ESPA +) survey data. We created two analysis samples by applying a propensity score matching method. The first sample consisted of women who received ANC at health facilities with guidelines matched with those who received ANC at health facilities without guidelines. The second sample consisted of women who received ANC from the providers who had undertaken in-service training in the last 24 months matched with women who received ANC from untrained providers. The outcome variable was the number of obstetric danger signs described during ANC counselling, ranging from zero to eight. The covariates included women’s socio-demographic characteristics, obstetric history, health facility characteristics, and ANC provider characteristics.We found that counselling women about obstetric danger signs during their ANC session varied according to the availability of ANC guidelines (61% to 70%) and provider training (62% to 68%). After matching the study participants by the measured covariates, the availability of ANC guidelines at the facility level significantly increased the average number of obstetric danger signs women received during counselling by 24% (95% CI: 12–35%). Similarly, providing refresher training for ANC providers increased the average number of obstetric danger signs described during counselling by 37% (95% CI: 26–48%).The findings suggest that the quality of ANC counselling in Ethiopia needs strengthening by ensuring that ANC guidelines are available at every health facility and that the providers receive regular ANC related in-service training.The online version contains supplementary material available at 10.1186/s12978-022-01442-6. Maternal death from preventable pregnancy-related complications remains a global health challenge. In 2017, there were 295,000 maternal deaths worldwide, and about two-thirds of these deaths were from Sub-Saharan Africa. Ethiopia is a Sub-Saharan African country with 401 maternal deaths per 100,000 live births in 2017, and this rate is higher than the target indicated in sustainable development goals. Most maternal deaths are due to obstetric complications and could have been averted through early detection and treatment. Providing antenatal care counselling about obstetric danger signs enhances women’s awareness of obstetric complications and encourages women to seek treatment from a skilled care provider. However, most women from low-income settings, including Ethiopia, do not receive counselling about obstetric danger signs. Facility-level antenatal care guidelines and provider in-service training improve antenatal care counselling. In Ethiopia, little is known to what extent antenatal care guidelines and provider training increase counselling on obstetric danger signs. The present study used the 2014 Ethiopian service provision assessment data and estimated the effect of antenatal care guidelines and provider training on counselling about obstetric danger signs. The analysis involved a propensity score matching method and included 1725 pregnant women. The study found that antenatal care guidelines at health facilities and antenatal care provider in-service training significantly increase counselling on obstetric danger signs by 24% and 37%, respectively. The finding suggests improving the quality of antenatal care counselling in Ethiopia needs antenatal care guidelines at each antenatal care clinic and refresher training for the providers.The online version contains supplementary material available at 10.1186/s12978-022-01442-6. Maternal death, the death of a woman while pregnant, during childbirth, or within 42 completed days of termination of pregnancy , remainsBetween 2000 and 2017, there was a 38% decline in the global maternal mortality ratio (MMR). In 2017 there were 295,000 maternal deaths, with an estimated 211 maternal deaths per 100,000 live births. This rate is higher than the SDG target. About 94% of these maternal deaths were from low-income countries. Sub-Saharan Africa (SSA) alone had 196,000 maternal deaths, accounting for nearly two-thirds of the global maternal deaths . As a reMost maternal deaths are preventable through quality healthcare services, such as antenatal care (ANC) . For exaStudies showed that ANC counselling, an interaction between the ANC provider and a woman and her family involving the exchange of information providing support , regardiFactors relating to a woman’s demand for health care and the supply of health services can influence the quality of ANC counselling. Examples of demand-related factors include a woman’s educational level  and the The importance of ANC guidelines and provider in-service training in improving ANC counselling on obstetric danger has been reported in studies from Benin , GuatemaThis is a cross-sectional study based on the 2014 Ethiopian service provision assessment plus (ESPA +) survey data . The 201The 2014 ESPA + survey data involved 1902 women receiving ANC at 1237 health facilities . We exclTo illustrate the relationship between ANC guidelines and ANC provider training, quality counselling and its outcome, we constructed a conceptual framework see Fig.  based onAny other symptoms or problems the client thinks might be related to this pregnancy” to address if each pregnant woman received counselling on any other pregnancy-related problems. A score of “1” was assigned to each obstetric danger sign if a woman was counselled; otherwise “, 0”. Thus, the counselling score ranges from 0 to 8. The score represents the number of obstetric danger signs counselled for a woman [Counselling on obstetric danger signs for pregnant women during ANC was an outcome variable measured using data from an observation checklist that recorded if the ANC provider counselled each pregnant woman on each obstetric danger. The 2014 ESPA + survey observation checklist included seven obstetric danger signs . In addition, the checklist used the statement “ a woman . FacilitGiven the non-experimental nature of the 2014 ESPA + survey data, the analysis involved a propensity score matching (PSM) method . IntroduWe calculated propensity scores for each treatment variable using Stata user-written command (psmatch2) . We used. We iteratively included the covariates in the propensity score model until we achieved an acceptable level of balance [Firstly, we identified the covariates to estimate the propensity score. The covariates included in the propensity score model were grouped into four categories: women’s socio-demographic characteristics,  obstetri balance , 56 and  balance , 59.Secondly, we calculated the standardised difference in proportions and means to assess whether propensity score matching has removed the differences in observed baseline characteristics between treated and control groups . We chosLastly, to determine the effects of treatment variables on the outcome variable, we chose the nearest neighbour one-to-one matching without replacement. The caliper distance of the propensity scores between the treated and control groups was set to be 0.01 and 0.002 for national ANC guideline and ANC provider training, respectively. While there is no consensus on the value of caliper distance , we deciOf the 1725 pregnant women included in the analysis, 713 (41.5%) attended ANC at health facilities with national ANC guidelines, and 815 (47.3%) received ANC service from a provider who took ANC related in-service training in the last 24 months preceding the ESPA + survey 2014.Table The nearest one-to-one matching without replacement yielded 1,274 samples comprised of 637 women who received ANC at health facilities with national ANC guidelines (treated) and 637 women who received ANC at health facilities with no national ANC guideline (control).We found that the absolute value of the calculated standardised difference in means and proportions of each variable after matching is less than 10%, indicating the presence of a balanced match on the observed baseline characteristics between treated and control groups.Figure After matching the study participants by the observed baseline characteristics, we found that facility-level ANC guidelines increased the average number of obstetric danger signs included in counselling women by 24% (95% CI: 12%—35%) , support to enhance quality infrastructure, and community participation , 82, 86.Applying the PSM method in an observational data (the 2014 ESPA + survey data) enabled us to estimate the unconfounded treatment effects of ANC guidelines and provider in-service training on the provision of counselling on obstetric danger signs. However, this study has some limitations. The PSM method only adjusts for measured covariates. Therefore, this study does not guarantee the elimination of bias due to unmeasured covariates. The PSM also excluded unmatched samples that may be systematically different from the matched samples, which could affect the representativeness of the study population. We acknowledge the findings in this study may or may not directly represent the current ANC counselling practice in Ethiopia because the 2014 ESPA + survey was undertaken seven years ago as of 2021. Thus, care should be taken to interpret the results. However, as the 2014 ESPA + survey is a national representative data, the findings of this study are comprehensive to provide policymakers with quality insights to improve ANC quality. Additionally, the providers’ uptake of in-service training was self-reported, which might have been affected by the recall and social desirability biases. Similarly, this study did not examine whether ANC providers consistently used the ANC guidelines during ANC counselling.We found that counselling pregnant women about obstetric danger signs during their ANC contacts is not universally practised. Nearly one in three women do not receive counselling on any obstetric danger sign. The availability of facility-level national ANC guidelines and in-service training for the ANC providers are positively and significantly associated with the number of obstetric danger signs discussed with women during ANC counselling. Decision-makers need to prioritise funding and policy to build supportive environments to ensure each health facility has guidelines and continuous in-service training programs are available for every ANC provider. Further research is required to understand whether ANC guidelines are consistently used during ANC service provision and examine whether this is associated with the quality of ANC counselling.Additional file 1. Figures illustrating the presence of match onobserved covariates between treated and control groupsbefore and aftermatching for both treatment variables (ANC guidelines andANC providers’training).


Conduction system pacing (CSP) encompassing His bundle (HBP) and left bundle branch area pacing (LBBAP) is gaining increasing attention in the electrophysiology community. These relatively novel physiological pacing modalities have the potential to outperform conventional pacing approaches with respect to clinical endpoints, although data are currently still limited. While HBP represents the most physiological form of cardiac stimulation, success rates, bundle branch correction, and electrical lead performance over time remain a concern. LBBAP systems may overcome these limitations. In this review article, we provide a comprehensive overview of the current evidence, implantation technique, device programming, and follow-up considerations concerning CSP systems. Moreover, we discuss ongoing technical developments and future perspectives of CSP. Implantable cardiac pacemakers (PMs) have been the mainstay for the treatment of bradyarrhythmia for decades. While these devices undoubtedly represent a unique success story since their first implantation in 1958, PMs have evolved significantly during the past years. With the introduction of leadless devices and conduction system pacing, the major limitations of conventional systems such as transvenous leads or unphysiological myocardial stimulation have been addressed. Conduction system pacing systems in particular have introduced a large variety of pacing options, which allow for tailoring antibradycardia stimulation individually to the patient’s needs. On the other hand, implantation complexity has increased considerably, and novel follow-up challenges have emerged. The goal of this review is to provide a state-of-the-art overview of conduction system pacing techniques, specific challenges, and clinically important implantation and follow-up tips.Conduction system pacing (CSP) encompasses His bundle pacing (HBP) and left bundle branch area pacing (LBBAP). The general rationale of CSP is to maximally benefit from the heart’s intrinsic conduction system to stimulate the myocardium in the most physiologic and efficient way possible.HBP was already performed more than 20 years ago in humans . ShortlySoon after the first HBP system implantations, acute beneficial hemodynamic and echocardiographic effects compared to conventional right ventricular (RV) pacing were demonstrated. Ventricular activation time was shortened during selective and non-selective HBP compared to RV pacing . HBP alsIn CRT candidates, HBP leads to an improved NYHA functional class by QRS shortening and improvement of the LV ejection fraction . Two ranHBP is the most physiologic pacing strategy, although the technique suffers from major limitations. The significant learning curve during the first 50–100 lead implantations needs to be overcome first . ReporteLBBAP comprises true left bundle branch pacing (LBBP), left bundle fascicular pacing (LBFP), and left ventricular septal pacing (LVSP). Definitions and distinctions between these modalities, however, are not uniformly used and differ amongst studies. Therefore, caution is warranted when comparing different trials.Compared to HBP, data on LBBAP are even more scarce and largely based on  registry studies. In general, capture thresholds are low and remain stable over time, and significant tricuspid valve regurgitation is rare . PatientPatients with a conventional CRT indication may benefit from LBBAP as well. In a large observational study that included 477 CRT and CSP patients , the paced QRS duration was shorter in LBBAP systems (133 vs. 153 ms). Echocardiographic response rates were greater in patients receiving a CSP system compared to patients with conventional CRT. In patients with LBBAP, LVEF improved from 26 to 41% compared to 26 to 34% in the conventional CRT group . These pSuccess rates of LBBAP lead implantation attempts are >90% for conventional bradycardia indications and >80% for heart failure indications according to a European multicenter registry (MELOS) . HoweverCSP lead implantation differs from traditional lead implantation regarding several key aspects and is more challenging. High-resolution fluoroscopy, a 12-lead ECG, dedicated implantation material, and trained implanters are required for CSP lead implantation. The introduction of three-dimensional (3D) guiding sheaths allows for better reach of the target area and has boosted interest in CSP in the past few years.Dedicated 3D guiding sheaths for CSP are available in various dimensions from Medtronic , Biotronik , Boston Scientific , and Abbott . They all require a 7–9F introducer sheath and provide a side-port for contrast delivery to perform septography. The sheaths facilitate reaching a suitable septal target site and provide additional backup, which may be of particular importance for LBBAP lead implantation, as the lead needs to penetrate deep into the interventricular septum. Some sheaths are deflectable or even equipped with mapping electrodes, which may be helpful for mapping the His bundle. The guiding sheaths are inserted using a J-wire at the beginning of the implantation and are slit at the end of the procedure.A lumen-less lead  with a 4.1F lead body and a 1.8 mm long fixed helix has initially been used for HBP in particular. However, for LBBAP, conventional stylet-driven leads  have been used for CSP as well ,30. The Sterile cables with crocodile clamps allow for connecting the CSP lead to the electrophysiology system or a pacing system analyzer (PSA). This is essential for (pace) mapping, repetitive impedance measurements, and standard lead measurements.The target site for HBP is fibers of the specific conduction system in the right atrium and the penetrating bundle, which is approximately 2–8 mm long and 1 mm thick. The HBP lead is advanced through the guiding sheath while mapping the target area in a unipolar fashion until a His bundle potential can be recorded. The lead is fixated by  4–8 clockwise turns of the whole lead. Selective or non-selective pacing (with correction of a left bundle branch block in the case of HBP in lieu of CRT) is ensured by uni- and bipolar pacing from the HBP lead. If correction of the QRS cannot be achieved, a His-optimized CRT (HOT-CRT) system with the additional implantation of a CS lead may be a more effective option, resulting in complex programming of the device. Distinguishing non-selective HBP from RV septal pacing may be challenging, thus, programmed stimulation and cutoff values for QRS duration, R-wave peak time in V6, and morphological criteria may help to identify the true His bundle capture ,32. PaciThe target implantation area for an LBBAP lead is significantly larger  but the CSP device programming and follow-up are challenging for several reasons. Up to now, there have been no designated devices in the market addressing CSP programming requirements. The CSP lead can be connected to the atrial, RV, or LV ports of the PM, which increases the overall complexity of CSP programming considerably. In addition, device follow-ups are more time-consuming and require a 12-lead ECG for threshold testing. The CSP lead may pace multiple structures simultaneously . The proper identification of the individual thresholds of these structures is crucial for tailored programming of CSP systems .HBP systems may suffer from poor CSP lead performance , which makes programming His bundle PMs often cumbersome and challenging . MoreoveIn contrast, programming LBBAP systems is relatively simple. The LBBAP lead is usually the only ventricular lead since a backup lead is rarely used and an additional coronary sinus lead does not provide better LV activation in the case of true LBB stimulation. For resynchronization, as in HOT-CRT, a CS-lead may be added if full recruitment of the left bundle cannot be achieved, resulting in a left-optimized CRT (LOT-CRT).In Currently, published studies investigating CSP are often monocentric, retrospective, and descriptive in nature. Randomized studies investigating CSP approaches are scarce, but numerous trials have been initiated. Amongst these, only some studies are briefly introduced here. The multicentric PROTECT-HF study aims to randomize 2600 patients with bradycardia pacing indication and a mildly reduced LV ejection fraction to either RV pacing or CSP (HBP or LBBAP) with the primary composite endpoint of death and heart failure hospitalizations. The smaller LEAP-BLOCK study is a randomized controlled trial designed to investigate whether LBBAP reduces the risk of RV-pacing-induced cardiac dysfunction compared to conventional pacing in patients with an AV block and normal LV function.Randomized trials comparing CSP and CRT systems have also been initiated but encompass only slightly more than 100 patients each. The CONSYST-CRT trial compares both modalities in patients with a class I or IIa CRT indication in a randomized non-inferiority fashion. Resynchronization will be provided by a conventional coronary sinus or a CSP lead. A composite endpoint of mortality, cardiac transplantation, heart failure hospitalization, and insignificant improvement of the LV ejection fraction after implantation will be analyzed. The LEFT-BUNDLE-CRT trial is a similar analysis with 176 patients qualifying for CRT-P/CRT-D implantation. The primary non-inferiority outcome measure will be a positive response to resynchronization defined by a clinical score or LV end-systolic volume.While all these studies will be important to assess the future prospects of CSP, key limitations have to be considered at this early stage of CSP system evolution. The limited implantation success rate and the long learning curve for implanters will inevitably result in a significant amount of crossovers in randomized trials hampering intention-to-treat analyses. Moreover, non-uniform success criteria used for LBBAP will impair comparability amongst studies.The introduction of dedicated delivery systems for CSP leads has enabled operators to routinely implant CSP leads. However, the available systems are still far from perfect, and manufacturers are constantly improving CSP hardware.Delivery sheaths have improved significantly with the introduction of different lengths, curves, and braided tubes for variable stiffness along the sheath body. Nonetheless, the manufacturer’s sheath portfolios still lack valuable tools .Future PM generators will offer the possibility to select the conduction system as a pacing site. Nominal programming values will be suggested accordingly, facilitating device programming. Moreover, improved dedicated CSP lead designs are under development. Lead prototypes include various designs such as fixed-screw leads offering a longer screw and a tempered tip design for better septal penetration.Finally, merging leadless technology and conduction system pacing approaches may be a highly desirable form of pacing in the future. Leadless systems have evolved rapidly during the past years and novel VDD systems provide relatively reliable AV synchrony . True duConduction system pacing is becoming increasingly popular. Novel hardware solutions provided by manufacturers and dedicated training courses for implanters facilitate the adoption of this new technique. However, despite the general enthusiasm CSP faces nowadays, structured evidence from randomized trials is still limited. Data are very encouraging but whether or not CSP will be the new Holy Grail for antibradycardia pacing remains to be investigated further. Thus, despite an increasingly liberal implantation practice, patient-specific counseling and case-by-case decisions seem advisable.