The coronavirus disease 2019 (COVID-19) creates a significant burden on the cardiovascular system. Moreover, diagnosing coronary artery disease in patients with COVID-19 may be clinically challenging. Herein, we present a case of in-hospital stent thrombosis and thrombotic occlusion of the right coronary artery after initial revascularization and adherence to antiplatelet therapy. The novel coronavirus disease 2019 (COVID-19) infection has been associated with complications secondary to coronary artery disease (CAD), with ST-elevation myocardial infarction (STEMI) being the most serious as it is associated with a significantly higher rate of in-hospital mortality [We present a case of a 48-year-old male who initially presented to the hospital with a chief complaint of pleuritic chest pain accompanied by fevers, dry cough, and headache for several days. The patient had a medical history of insulin-dependent diabetes mellitus, hypertension, and mixed hyperlipidemia. He is also a former smoker. On arrival, his vital signs revealed a blood pressure of 118/62 mmHg, pulse of 95 beats/minute, breathing rate of 20 breaths/minute, and oxygen saturation of 96% while breathing ambient air. Initial electrocardiography (EKG) showed ST-segment elevations in leads II, III, and V2-V5 Figure . ThrombeDiagnosing STEMI in patients with COVID-19 has proved challenging, and reports have shown that in patients with COVID-19 presenting with STEMI, a culprit lesion is not always present . BesidesIntriguingly, stent thrombosis has been associated more frequently with first-generation stents . Acute sCOVID-19 associated coronary artery thrombotic events carry a significant burden on diagnosing and managing such a condition. STEMI can be challenging to diagnose in patients with COVID-19, as neither EKG changes nor elevated troponin is consistent with obstructive CAD. A multidisciplinary approach should be initiated when dealing with a myocardial injury in patients with COVID-19. To the best of our knowledge, PCI remains the recommended modality when dealing with STEMI in patients with COVID-19. However, recurrent thrombotic events following revascularization merit further assessment of this modality when dealing with this patient population.


Evolut™ R, Medtronic, Dublin Ireland).We describe one of the first-in-human cases of valve-in-valve transcatheter aortic valve implantation (TAVI) using fluoroscopic-only guidance for the treatment of late failure of a bio-prosthetic Bentall conduit, the BioValsalva™ Vascutek , using a self-expandable heart valve prosthesis (CoreValve™ Over the past decade, transcatheter aortic valve implantation (TAVI) has emerged as a valid and safe treatment option for severe symptomatic aortic stenosis, from prohibitive surgical risk patients in the early days to those at low-to-intermediate risk in current practice -6.Advances in device technology, as well as growing operators’ skills, were remarkable in this short time window. This set the stage for a fantastic expansion of the procedure indications towards increasingly challenging clinical scenarios. Valve-in-valve TAVI has become a safe and reproducible procedure for patients with severe bioprosthetic valve dysfunction deemed to be at high risk for re-operation.Pure (native or prosthetic) valve regurgitation is still a grey zone, and evidence in this challenging subset is much needed. These patients typically exhibit less annular and/or leaflet calcifications, implying technical difficulties in adequate transcatheter aortic valve anchoring. Furthermore, non-calcified native aortic valves and non-radiopaque bioprosthesis both challenge correct positioning under fluoroscopic guidance.We describe one of the first in-human cases of valve-in-valve TAVI using fluoroscopic-only guidance for the treatment of late failure of a bio-prosthetic Bentall conduit, the BioValsalva™ Vascutek , using a self-expandable heart valve prosthesis .The patient was a 65-year-old woman with no known cardiovascular risk factors. Relevant past medical history included epilepsy and primary biliary cirrhosis. She was submitted to a Bentall surgical procedure, with implantation of a 25mm porcine bioprosthetic valved conduit  in 2008, following documentation of bicuspid aortic valve with severe regurgitation and a dilated ascending aorta.She was chronically medicated with venlafaxine, levetiracetam, pantoprazole, aspirin, furosemide, and bisoprolol.The patient presented with new-onset heart failure symptoms twelve-years after the Bentall procedure, New York Heart Association (NYHA) III functional class, and severe transprosthetic aortic regurgitation was diagnosed. She was refused for re-do cardiac surgery considering high surgical risk - the calculated EuroScore II was 18.52%, and the Society of Thoracic Surgeons (STS) score for mortality was 8.60%.Procedural planningTransoesophageal echocardiogram showed thickness and retraction of the valve cusp positioned in the right coronary position and a prolapse of the cusp in the non-coronary position resulting in severe, eccentric, transprosthetic aortic regurgitation. There was also a mild enlargement of the left cardiac chambers (indexed left ventricular end-diastolic volume of 68ml/m2), and left ventricular systolic function was mildly depressed (left ventricle ejection fraction of 49%). Coronary artery disease was excluded by invasive angiography. A pre-procedure CT scan is shown in figure Detailed aortic annulus and aortic root measurements are shown in Table TAVI procedureThe procedure was performed using right transfemoral access, under fluoroscopic guidance. Baseline aortography .A recently published meta-analysis comprising 594 patients from six observational studies, of whom 255 underwent valve-in-valve (ViV) TAVI and 339 underwent redo surgical aortic valve replacement (SAVR), reiterated the safety and feasibility of ViV-TAVI for failed aortic bio-prosthetic valves in patients deemed to be at high risk for surgery . In thisData from the Global Valve-in-Valve Registry showed that bioprosthetic valve failure might present as predominant regurgitation in up to 34% of patients, as a consequence of wear and tear, calcification, or infection . ViV-TAVAnother group reported successful treatment of a failed BioConduit with severe regurgitation, using transoesophageal echocardiographic guidance . ViV TAVThis is, to the best of our knowledge, one of the first literature reports of a valve-in-valve TAVI for the treatment of late failure of a BioValsalva Vascutek conduit with severe aortic regurgitation, using the CoreValve device. The patient had an uneventful recovery in the early postoperative period and was discharged after four days. With this challenging case, the authors show the feasibility and safety of ViV-TAVI in this innovative scenario, when performed in high-volume centers and by experienced operators.


Cancer heterogeneity and progression are subject to complex interactions between neoplastic cells and their microenvironment, including the immune system. Although glioblastomas (GBMs) are classified as ‘cold tumours’ with very little lymphocyte infiltration, they can contain up to 30–40% of tumour-associated macrophages, reported to contribute to a supportive microenvironment that facilitates tumour proliferation, survival and migration. In GBM, tumour-associated macrophages comprise either resident parenchymal microglia, perivascular macrophages or peripheral monocyte-derived cells. They are recruited by GBMs and in turn release growth factors and cytokines that affect the tumour. Notably, tumour-associated microglia/macrophages (TAMs) acquire different expression programs, which shape the tumour microenvironment and contribute to GBM molecular subtyping. Further, emerging evidence highlights that TAM programs may adapt to specific tumour features and landscapes. Here, we review key evidence describing TAM transcriptional and functional heterogeneity in GBM. We propose that unravelling the intricate complexity and diversity of the myeloid compartment as well as understanding how different TAM subsets may affect tumour progression will possibly pave the way to new immune therapeutic avenues for GBM patients. Gliomas represent approximately 80% of all malignant tumours of the central nervous system (CNS) . Among tThe immune landscape of brain tumours is intensely investigated, unveiling new insight in the interactions between neoplastic cells and the immune system . GBM is Due to their large number in the tumour microenvironment, TAMs represent a key target for GBM immunotherapy and a range of immunomodulatory agents are currently being trialled in patients. For example, as TAMs critically depend on colony-stimulating factor-1 (CSF-1) for their survival, differentiation and proliferation, strategies to target TAMs in the clinic include CSF-1 receptor (CSF-1R) blockade ,8. HowevDespite extensive efforts in this direction, the precise role of TAMs in GBM onset and progression as well as how TAMs may affect current immunotherapeutic approaches, including vaccines, oncolytic viruses and immune-checkpoint inhibitors, remains unclear. Therefore, a deeper understanding of the complexity and diversity of TAM adaptive features is critical to develop novel personalized immune therapeutic strategies for GBM patients.In this review, we will describe different features underlying TAMs heterogeneity and adaptation in GBM. Further, we will pinpoint the aspects linked to their diversity that warrant further investigations and how this heterogeneity may ultimately be harnessed for the development of novel personalized immune therapeutic strategies.Macrophages are highly dynamic cells whose molecular profiles are substantially influenced by specific environmental cues. In vitro studies enabled classification of activated macrophages according to a binary system, with pro-inflammatory cytokines  skewing them towards a classical (M1-like) activation state, while anti-inflammatory cytokines  polarizing macrophages into an alternative (M2-like) phenotype . A similThe healthy brain harbours specific populations of tissue-resident macrophages effectively located in the parenchyma, perivascular spaces, meninges and choroid plexus where they maintain tissue homeostasis and ensure immune functions . Within In GBM, TAMs are recruited to the tumour site through various mediators, including CCL2, CX3CL1, CSF-1, GM-CSF and osteopontin released by neoplastic cells ,27,28,29+CD45low cells) from peripheral monocyte-derived macrophages (CD11b+CD45high cells) [Itga4 (CD49D), enabling the distinction between microglia and monocyte-derived macrophages in murine tumours [P2ry12, Tmem119, Slc2a5 and Fcrls, whereas Emilin2, Gda, Hp and Sell were mainly expressed by peripheral monocytes/macrophages [Experimentally, approaches to distinguish resident microglia from other inflammatory immune cells entering the CNS have traditionally relied on CD45 expression to discriminate resident microglia (CD11bh cells) . Howeverh cells) . Recentl tumours   signalling pathways in microglia reducing their phagocytic and inflammatory responses . In cancTo date, the combination of radio-chemotherapy with immunotherapeutic agents has not been effective in GBM and drugs driving anti-tumour immune responses are currently evaluated in clinical trials. In principle, radiation can increase in situ immunogenicity of malignant cells, thus improving tumour immune recognition and T-cell mediated anti-tumour responses . In thesCcl2 genetic ablation, prolonged the survival of tumour-bearing mice [+ population and contributed to 30% of tumour increase in the GBM syngeneic GL261 mouse model [Upon accumulation to the tumour site, TAMs are thought to drive immune-suppression and promote tumour progression. Due to their high numbers in GBM, their genomic stability and adaptability to the microenvironment, several strategies to deplete TAMs have been developed. For example, liposome-encapsulated clodronate, which has been commonly used to deplete macrophage populations by inducing their apoptosis once phagocytosed by the cells, reduced tumour invasion in GL261 cultured brain slices, which was restored after addition of TAMs . Howevering mice , but these model . A majorse model . Furtherse model .These results highlight that depleting TAMs indiscriminately is probably not the optimal approach, as TAMs might play different roles depending on GBM features, including immunogenicity.Immunotherapy is emerging as a promising approach holding great potential to foster tumour elimination by unleashing the immune system. The intense crosstalk between tumour cells, antigen presenting cells (APCs) and T cells is intricately controlled by multiple ligand-receptor interactions, known as checkpoints, which generally inhibit T-cell activation, ultimately affecting T cell cytotoxicity against tumour cells . For exaCcr2RFP) and microglia (Cx3cr1GFP), in which microglia were found to effectively phagocytose tumour cells in response to anti-CD47 blockade with a reduced inflammatory signature, making them a promising target for clinical applications [Due to TAMs abundance within GBM and their fast response to external stimuli, strategies to re-educate TAMs in mouse glioma models may be more efficient than their depletion or the use of immune checkpoint inhibitors. In this context, a promising target is signal-regulatory protein (SIRP) α, an inhibitory receptor expressed on myeloid cells that recognizes the CD47 ligand on tumour cells and contributes to immune evasion. The targeting of this axis with humanized anti-CD47 antibodies enhanced tumour phagocytosis and reduced tumour burden in patient-derived orthotopic xenografts of paediatric brain tumours . Interesications . Anotherications .+ regulatory T cells as well as accumulation of tumour-infiltrating effector T cells exhibiting anti-tumorigenic features [Combinatorial approaches targeting immune-suppressive populations concomitantly with promoting endogenous anti-tumour immune responses successfully impaired tumour progression in various subcutaneous tumour models. For example, dual targeting of suppressive myeloid populations by inhibiting CSF-1/CSF-1R signalling and activation of APCs with CD40 agonists conferred superior anti-tumour efficacy and increased survival compared with monotherapy. This effect was attributed to the decrease of immunosuppressive TAMs and Foxp3features . Furtherfeatures . Lastly,Taken together, strategies aiming at reprogramming immunosuppressive myeloid cell populations and/or fostering anti-tumour immune responses in the tumour microenvironment may be necessary to empower checkpoint-based immune therapeutics in GBM. However, TAMs heterogeneity may represent a barrier to non-selective immunotherapies, which seek to target TAMs indiscriminately.In the last 10 years, multiple attempts have used transcriptional profiling to sub-classify GBMs into clinically meaningful tumour subtypes ,60,61. ARecent scRNA-seq studies also highlighted tissue-specific myeloid cell heterogeneity associated with distinct brain region-dependent transcriptional identities in health and disease ,67,68. FIL1α and IL1β) were highly expressed at the tumour periphery, while a more anti-inflammatory phenotype  was observed in the tumour core  and alternatively activated (M2) genes in individual cells  Figure . Furtherour core . Lastly,ignature .The cellular composition of IDH-mutant gliomas was also unveiled by scRNA-seq, suggesting that astrocytomas (IDH-A) and oligodendrogliomas (IDH-O) share common lineages of glial differentiation with distinct tumour microenvironment signatures . SpecifiIn IDH-wildtype gliomas, it has been very recently shown that TAMs acquire a disease-associated signature related to aging microglia programs, including downregulation of the microglia homeostatic genes and upregulation of inflammatory, metabolic and interferon-associated genes. Various TAM clusters, including subsets enriched for positive regulation of vasculature development or antigen processing via MHC class I, have been identified  Figure . Taken tMicroglia and macrophages in GBM are educated by the tumour and display unique molecular programs, which largely drive tumour-supportive phenotypes. However, the available subpopulations and functions of these cells along GBM development and progression are only partially understood. TAMs have been classified based on their activation state, their function and their morphology. With the advent of single cell analyses, it has become increasingly clear that the classification is complex and does not fully capture the heterogeneity of these cells in the context of GBM. Immunotherapeutic approaches in GBM will need to take into account the role of TAMs and their functional, spatial and temporal heterogeneity.The local GBM microenvironment actively reprograms TAMs to establish new functional states with distinct gene expression profiles. If so, which TAM subsets arise during GBM development? Which subset of TAMs are more prone to be re-educated? Are their dynamic molecular states associated to TAM specific functions along GBM development and progression? Will their dissection help to improve TAM targeted therapies in combination with current treatment regimens? Taken together, it will be critical to address these questions to determine the most appropriate combinatorial approaches and to identify patient subgroups that may benefit most.As perspectives, it will be fundamental to combine single-cell approaches, such as scRNA-seq and imaging mass cytometry, with functional screening of inferred cellular diversity, which will be critical to identify TAM subsets across GBM subtypes, landscapes and tumour stages, thus enabling targeting of putative pro-tumorigenic TAM subpopulations and/or to empower the anti-tumorigenic ones. Further, shedding light on the functional crosstalk between neoplastic cells and the tumour microenvironment at single-cell resolution will enable to dissect complex cell–cell interactions and how these may affect patient outcomes. Using a combination of scRNA-seq and flow cytometry in syngeneic mouse models of solid tumours allowed to profile potential cell-cell interactions between neoplastic and non-neoplastic cells . Buildin


Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is a threat to cassava production in Africa. The potential spread of CBSD into West Africa is a cause for concern, therefore screening for resistance in farmer-preferred genotypes is crucial for effective control and management. We multiplied a selection of eleven cassava cultivars grown by farmers in Ghana to test their response to a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates using a stringent top-cleft graft inoculation method. Virus titers were quantified in the inoculated scions and cuttings propagated from the inoculated scions to assess virus accumulation and recovery. All cultivars were susceptible to the mixed infection although their response and symptom development varied. In the propagated infected scions, CBSV accumulated at higher titers in leaves of eight of the eleven cultivars. Visual scoring of storage roots from six-month-old virus-inoculated plants revealed the absence of CBSD-associated necrosis symptoms and detectable titers of CBSVs in the cultivar, IFAD. Although all eleven cultivars supported the replication of CBSV and UCBSV in their leaves, the absence of virus replication and CBSD-associated symptoms in the roots of some cultivars could be used as criteria to rapidly advance durable CBSD tolerance using breeding and genetic engineering approaches.Cassava brown streak disease (CBSD) caused by the  Manihot esculenta Crantz) is an important source of carbohydrate in the diets of nearly 800 million people, one-third of whom live in sub-Saharan Africa (SSA) . In-depth characterization of leaf and root CBSD symptom development, virus replication as well as the impact of vegetative propagation on viral disease transmission is reported.Molecular screening of leaf material from eleven field-grown cassava cultivars obtained from the BNARI germplasm collection in Ghana  and leafTogether, our results indicate that the eleven selected cassava cultivars from Ghana obtained from the BNARI germplasm collection were free of CBSVs and that CBSVs are currently absent from the surveyed farmers’ fields. However, extension of molecular screening to cassava fields across cassava-growing regions in Ghana will be important to confirm the absence of CBSVs and inform CBSD prevention strategies.The top-cleft graft method has proven to be the most stringent method to inoculate cassava with CBSVs as demonstrated by the reported 100% transmission rates for CBSV and UCBSV ,28,31,32To test the response of the selected farmer-preferred cultivars to CBSD, we used Ebwanateraka cassava rootstocks carrying a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02). We previously showed that Ebwanateraka rootstocks support high levels of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) replication ,31,32. TA 100% infection incidence A with ty. For example, even though Afisiafi had the lowest mean CBSD incident and leaf symptom severity (In order to determine the accumulation levels of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) in the selected cassava cultivars, we measured viral titers in top-grafted scions as well as in cuttings propagated from inoculated scions using RT-qPCR A. Both Cseverity , it accuseverity B. NotablVirus disease management strategies in cassava have exploited the phenomenon of reversion, which is the production of virus symptom-free plants from infected parent plants . ReversiCBSV is reportedly more aggressive than UCBSV ,19,42, aTo evaluate the effect of CBSD on the viability of clonal stem cuttings propagated from infected scions of the selected Ghanaian cultivars, we multiplied a minimum of nine stem cuttings per cultivar (three nodes per cutting) from CBSD-infected scions and grew them under greenhouse conditions. Compared to CBSD non-infected cuttings, the survival rates of sprouted cuttings were reduced in all eleven Ghanaian cassava cultivars after CBSD infection. Specifically, less than 50% of cuttings propagated from infected scions failed to sprout except in Bosomnsia, Megyewontem and Nkabom, which had a higher percentage of cuttings that sprouted. For example, in Afisiafi, which was classified as tolerant based on its ability to restrict CBSD symptom expression and severity, the accumulation of high levels of CBSV titers in the inoculated scions might explain the reduced survival rates of Afisiafi cuttings. Comparatively, between 78 to 100% of cuttings propagated from non-infected Ghanaian cultivars sprouted by 12 weeks in the greenhouse .It has been previously reported that endemic CBSV isolates are associated with severe symptoms and significant reduction in the sprouting of cuttings compared to milder epidemic isolates ,42. The To analyze the accumulation of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) in storage roots of the cassava cultivars, stem cuttings were propagated from CBSD-infected scions and grown for 6 months under greenhouse conditions in order to produce storage roots. The storage roots were harvested and visually assessed for typical CBSD root necrosis symptoms. The eleven cultivars graft-challenged with rootstocks carrying a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) were subsequently propagated by stem cuttings  and UCBSV (TAZ-DES-02) isolates in leaf material from the farmers’ fields surveyed in four cassava-producing regions in Ghana is important because it will inform CBSD prevention strategies. The implementation of prevention strategies in a coordinated manner among the cassava community can limit the spread of CBSD into West Africa.Cuttings from field-grown plants of eleven farmer-preferred cassava cultivars, Ankra, Afisiafi, ADI 001, Bosomnsia, Dagarti, IFAD, Megyewontem, Nkabom, Santum, Tuaka and Tomfa were collected from the cassava germplasm collection of the Biotechnology and Nuclear Agriculture Research Institute (BNARI), Ghana. These cuttings were established in the greenhouse at ETH Zurich, Switzerland  and subsequently multiplied by stem cuttings. In addition, to survey the presence of CBSV in Ghana, eighty cassava leaf samples were collected from farmers’ fields in four cassava-growing regions , Eastern region , Ashanti region  and Greater Accra region ). Twenty samples were collected from the Greater Accra region, twenty samples from the Central region, ten samples from the Eastern region and thirty samples from the Ashanti region. Samples were stored at −80 °C until processed.The virus isolates used in this study were CBSV  and UCBSV . Both isolates were maintained in cassava genotype Ebwanateraka under greenhouse conditions at 27 °C and 60% relative humidity. Using a previously established top-grafting method  virus-frLeaves of CBSD challenged plants were visually assessed and scored for CBSD symptoms 12 weeks post grafting using a scoring scale where 1 = no visible CBSD symptoms, 2 = mild foliar symptoms on some leaves, 3 = pronounced foliar symptoms but no die-back, 4 = pronounced foliar symptoms with some die-back of terminal branches, and 5 = severe foliar symptoms and plant die-back ,41,42. ATotal RNA was isolated from 1 g of cassava leaves using the modified cetyltrimethyl ammonium bromide (CTAB) method . First sw/v agarose gel with images recorded using a Gel iX20 imager gel documentation system  to confirm the presence or absence of cassava brown streak viruses. The selected eleven Ghanaian cassava cultivars were screened for CBSVs prior to CBSD resistance challenge using degenerate primers designed to detect the coat protein of CBSVs including CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) viral isolates [For detection of CBSV and UCBSV, cDNA was diluted in a ratio of 1:3 (1 part of cDNA to 3 parts of water). A total volume of 4 µL of the cDNA template dilution mixture was used for RT-PCR. PCR thermal cycle conditions used were as follows: Initial denaturation for 20 s at 95 °C, 40 cycles of denaturation for 3 s at 95 °C, annealing for 15 s at 60 °C and extension for 30 s at 72 °C. PCR products were resolved on 1% isolates . Similar® Green Master Mix , 1 µL each of forward and reverse primers  and 4 µL of distilled water in a total volume of 20 µL. For each cultivar, we analyzed samples from three individual plants  with two technical replicates each using primers specific for CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) viral isolates [For quantitation of virus titers in leaf and storage roots of Ghanaian cultivars after CBSD graft-challenge, we performed RT-qPCR with a 7500 Fast Real-Time PCR System  using the ABI 7000 sequence detection system (SDS) for analysis. The RT-qPCR reaction mix consisted of 4 µL of cDNA template, 10 µL SYBRisolates ,28  and UCBSV (TAZ-DES-02) isolates, in field-grown cassava cultivars in Ghana is encouraging for cassava production at this time. Nonetheless, our results should also encourage extensive and continuous surveillance of CBSVs in farmers’ fields across all cassava-producing regions in Ghana.In addition, continued screening and characterization of cassava germplasm for responses to CBSD infection is important to identify genotypes with CBSD tolerance or resistance traits . The deployment of such tolerant or resistant cultivars can effectively control the spread and reduce losses associated with CBSD infection. It is worth noting that the time and challenge associated with conventional breeding of cassava for resistance to CBSD should also encourage other approaches. Implementation of transgenic technologies in farmer-preferred varieties can complement conventional breeding and speed up the development of durable resistance against CBSD in Africa ,55.


RET gene fusions in 1% to 2% of non–small-cell lung cancers (NSCLC) and by RET gene mutations in most medullary thyroid cancers (MTC).1 Although multikinase inhibitors (MKIs) with nonspecific anti-RET activity are approved for the treatment of MTC irrespective of RET mutation status, their investigational use for patients with RET fusion-positive NSCLC has been limited by substantial off-target adverse effects that lead to dose reductions and inadequate RET-specific inhibition.7 As a result, tumor responses to MKIs have been infrequent and short lived, and a comprehensive molecular understanding of MKI response and resistance is lacking.The RET receptor tyrosine kinase is oncogenically activated by 8 In an ongoing, phase I/II study of LOXO-292 , confirmed partial responses (PRs) were achieved in most RET-altered patients across different tumor histologies and diverse RET gene fusions and mutations. Moreover, activity has been seen after acquired resistance to systemic therapies, including MKIs, and in intracranial disease.11 Responses have been generally long lasting with the median duration of response not yet reached, and the safety profile has been tolerable, consistent with preclinical high selectivity.In contrast to the MKIs, LOXO-292 is a highly selective, small molecule RET tyrosine kinase inhibitor (TKI) with nanomolar potency against diverse RET alterations, favorable pharmacokinetic (PK) properties, and significant CNS penetration.12 Recently, we reported the emergence of a RET valine-to-methionine (V804M) mutation in circulating tumor DNA from a patient with RET-mutant, sporadic MTC treated previously with multiple MKIs, and the ability of LOXO-292 to induce a durable tumor response in this patient.13 Two other reports described the emergence of RET kinase domain mutations (RET V804M and RET S904F) each in a single patient with RET fusion-positive NSCLC during treatment with the anti-RET MKI vandetanib.15 Their overall frequency and clinical actionability are not clear.LOXO-292 was designed to overcome mutations in the RET V804 gatekeeper residue shown previously to cause preclinically acquired resistance to anti-RET MKIs.RET-altered cancers, one with acquired gatekeeper mutation resistance to an anti-RET MKI and the second with a germline gatekeeper mutation in the setting of multiple endocrine neoplasia 2A.Here, we describe the ability of LOXO-292 to overcome acquired RET kinase domain mutations preclinically and in two patients with KIF5B-RET fusion in a sample from a recurrent pleural fusion  1.1 with vandetanib treatment, with improvement in multiple lung and pleural-based lesions. She progressed after 13 months of treatment  gatekeeper mutation absent from a pretreatment tumor sample  calculated KIF5B-RET and KIF5B-RET-V804L target coverage at steady state , but this resistance may be overcome by next-generation inhibitors .14 To our knowledge, this is also the first published report of a patient with hereditary RET V804M-mutant MTC treated successfully with RET-targeted therapy. For both patients, integration of preclinical RET target inhibitory activity with the actual exposures safely achieved at the recommended phase II dose of 160 mg twice a day allowed more than IC90 calculated target coverage of both wild-type and gatekeeper-mutated RET in patients (To our knowledge, case 1 is the second patient with NSCLC to be described as having an acquired RET gatekeeper mutation after prior anti-RET MKI treatment and the first to be treated successfully with RET-targeted therapy.patients  and 2B. RET-altered cancer exposed previously to anti-RET MKIs.In conclusion, the highly selective anti-RET TKI LOXO-292 led to durable tumor responses in two patients with RET V804 gatekeeper mutations, one acquired and one germline. These results illustrate the importance of anticipating mechanisms of acquired resistance when developing new targeted therapies and underscore the potential of selective RET inhibition with LOXO-292 to treat patients with


To develop and investigate a deep learning approach that uses sparse-view acquisition in dedicated breast computed tomography for radiation dose reduction, we propose a framework that combines 3D sparse-view cone-beam acquisition with a multi-slice residual dense network (MS-RDN) reconstruction. Projection datasets  from 34 women were reconstructed using the FDK algorithm and served as reference. Sparse-view  projection data were reconstructed using the FDK algorithm. The proposed MS-RDN uses the sparse-view and reference FDK reconstructions as input and label, respectively. Our MS-RDN evaluated with respect to fully sampled FDK reference yields superior performance, quantitatively and visually, compared to conventional compressed sensing methods and state-of-the-art deep learning based methods. The proposed deep learning driven framework can potentially enable low dose breast CT imaging. A multi-reader, multi-case receiver operating characteristic (ROC) study employing 18 readers and 235 cases showed improved sensitivity of non-contrast diagnostic BCT over mammography-based diagnostic work-up3, leading to its regulatory approval for non-contrast diagnostic use. Non-contrast BCT can have a far greater role if its suitability for breast cancer screening is demonstrated. The radiation dose  from non-contrast diagnostic BCT, while similar to the MGD from mammography-based diagnostic workup, was approximately twice that of 2-view (standard) screening DM4. At radiation dose similar to mammography, a prior study using an early prototype showed improved visualization of masses and reduced visualization of microcalcifications with BCT compared to mammography5. Hence, the long-term goal is to reduce the radiation dose to be comparable to mammography screening, without loss of detection performance.Dedicated breast computed tomography (BCT) is an emerging, fully 3D, high-resolution (100–300 µm nearly isotropic voxels) imaging modality that does not employ physical compression of the breast. Compared to digital breast tomosynthesis7, low-noise, high-resolution, complementary metal oxide (CMOS) detectors9 and beam-shaping X-ray filters11 are being investigated. Acquisition strategies being investigated include helical scan6, laterally-shifted detector geometry13 short-scan14, and sparse-view acquisition15. Also, theoretical and empirical optimization of x-ray beam quality for acquiring projection data have been reported19.Radiation dose reduction in BCT to levels suitable for breast cancer screening can be achieved through improved hardware, acquisition strategies and advanced image reconstruction inclusive of post-processing techniques. In terms of hardware, photon-counting detectors22 and denoising techniques23 have been investigated for BCT, all BCT systems currently use Feldkamp–Davis–Kress (FDK) reconstruction24. Deep learning based image reconstruction has not been investigated in the context of BCT or for cone-beam CT; however, it has been explored for conventional multi-detector CT28. Jin et al.25 utilized the U-Net with residual learning and demonstrated the feasibility on parallel beam X-ray CT. A similar approach was independently proposed by Chen et al.26. The proposed residual encoder-decoder convolutional neural network (RED-CNN)26 was shown to be quantitatively outperforming the earlier version29 and the wavelet-domain CNN30.In this study, we describe the potential of advanced image reconstruction employing deep learning techniques that can be used with existing BCT technology. This can lead to lower radiation dose and expedite its translation for breast cancer screening. This study is complementary to ongoing hardware-oriented research. Although, statistical iterative reconstruction31 or dense blocks32 have shown improved performance compared to standard convolutional neural networks in computer vision applications34. In this work, we adopt a derived version of the residual dense network33 and investigate its potential for low-dose cone-beam BCT image reconstruction.Recently, advanced network architectures using residual blocks35 diagnostic assessment category 4 or 5, who had previously participated in an IRB approved, Health Insurance Portability and Accountability Act (HIPAA)-compliant research study . The study was conducted with informed consent from participants involved. This dataset was used in several prior studies41. All subjects underwent non-contrast dedicated breast CT exam of the ipsilateral breast using a clinical prototype flat-panel cone-beam breast CT system . The scan parameters were: 49 kVp, 1.4 mm of Al 1st HVL, 8 ms pulse-width, 300 projection views, 360 degree full-scan acquisition, 12.6 mGy MGD, and 10 s scan time. The 300 view projection datasets were reconstructed using the FDK algorithm with 0.273 mm isotropic voxel pitch and matrix size This retrospective study was conducted in accordance with relevant guidelines and institutional review-board (IRB) approved protocol . The study used de-identified projection datasets from 34 women assigned Breast Imaging-Reporting and Data System (BIRADS)26. Test subject datasets were reconstructed by the single-slice networks with and without TOI selection. FDK reconstructions on the 300-view data (denoted as FDK300) were used as references across all the experiments. The performance was quantitatively evaluated with Normalized Mean Square Error (NMSE), bias, Peak Signal-to-Noise Ratio (PSNR), and Structural Similarity Index Metric (SSIM42). All four metrics significantly differed across all reconstructions  and RED-CNNmentclass2pt{minimOver the entire test dataset, MS-RDN with mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimOur MS-RDN was compared with RED-CNN in three sets of network configurations: single slice training without TOI selection  based reconstruction framework for 3D sparse-view breast CT. In reference to full view FDK reconstructions, the proposed framework yields image quality superior to compressed sensing techniques such as FIRST while requiring comparable reconstruction times. In this study, the reconstructed FOV was relatively large  more similar to the FIRST approaches in terms of signal-to-noise ratio. Recent studies46 suggest that pixel-wise losses, such as 48, perceptual loss49, as well as texture matching loss50 are proven to preserve high frequency image content and improve the perceptual quality. However, it should be noted that these techniques may hallucinate high frequency textures44, which makes them less appealing for medical applications. In breast CT imaging, hallucinated high frequency texture may mimic microcalcifications. Nevertheless, the impact of alternative loss functions in dedicated breast CT needs to be investigated and can be an extension of the current work.Our DL-based framework uses residual dense blocksWe also investigated the possible failure cases for the proposed deep learning technique. For the example shown in Fig. 24. The reconstruction process can be expressed using the following equation52. Reconstructed volumes are assumed to have isotropic voxel resolution as the voxel sizes are principally determined by size of the imaging detectors. However, the spatial resolution can be location-dependent and anisotropic due to reduced sampling at the periphery of the field of view within a transverse slice and due to geometric distortions arising from cone-beam geometry (commonly referred to as cone-beam artifacts) as the acquisition does not satisfy data-completeness requirement54 with the exception of the central transverse slices.In 3D cone-beam BCT, multi-projection data 57. This sparse-view projection data was obtained by retrospectively undersampling the full-view projection data u denotes the undersampling factor, and the notation i: j: k in Eq. ,,3), resp70. Inspired by these techniques, patches that contain more than Figure Z, the ensemble strategy to obtaining the target slice reconstruction f denotes the ensemble function, t, and i of the input. In our experiment, we found evenly averaging is a simple yet effective ensemble approach. We replicate border slices to handle slices at edges.The network testing phase is illustrated in Fig. 44, our feature branch is sequentially composed of multiple dense compression units (DCUs)33, a 71. The DCU structure efficiently merges local feature information and periodically breaks dense connections to improve back projection of gradients33. Figure 32.The proposed MS-RDN architecture is shown in Fig. To demonstrate the superiority of multi-slice training, we first trained multiple MS-RDNs with the same configurations except for the number of adjacent slices, i.e., 26 designed for low dose CT image reconstruction. We followed the implementation of RED-CNN from https://github.com/SSinyu/RED_CNN and adopted the suggested network parameters . Note that unlike our proposed deep learning reconstruction framework, RED-CNN26 was trained with randomly extracted single-slice patches. We therefore applied the TOI selection and multi-slice training scheme to the RED-CNN architecture for comparison.Our MS-RDN was compared with the residual encoder–decoder convolutional neural network (RED-CNN)42). The NMSE metric was computed as the ratio of mean square error to mean square of the reference image and converted into decibel (dB), that isNine randomly selected test subjects were evenly grouped by the size of breast. To reduce the impact of breast size or slice location, we always select a constant number of measurement samples within the breast for quantitative analysis. The network reconstructions were evaluated with normalized mean square error (NMSE), bias, peak signal-to-noise ratio (PSNR), and Structural Similarity Index Metric (SSIM22) was also used for sparse-view image reconstruction. This algorithm is an ultra-fast variant of the adaptive steepest descent-projection on to convex sets (ASD-POCS72) and has been shown to suppress additional artifacts on the periphery of the object. The performance of FIRST was compared to MS-RDN using one small-size breast, one medium-size breast, and one large-size breast.The fast, iterative, tv-regularized, statistical reconstruction technique  were used to test if the metric  differed between the reconstructions, as the same set of test cases were reconstructed using different methods. Effects associated with Supplementary Information.


The coronavirus disease 2019 (COVID-19) pandemic outbreak, caused by severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) is affecting people worldwide representing a public health emergency. The effect of concomitant COVID-19 on patients who underwent cardiac surgery using cardiopulmonary bypass (CPB) is still undefined. Both SARS-Cov-2 infection and CPB can develop a cytokines storm and haemostatic disarrangements leading to acute respiratory distress syndrome (ARDS) and post-perfusion lung syndrome, respectively. SARS-Cov-2 infection may trigger and exacerbate post-inflammatory state after CPB resulting in higher risk of post-surgical adverse outcomes. International guidelines lack to provide standard management protocols for pre-operative COVID-19 patients requiring non-deferrable cardiac surgery intervention. We present a report of a successful coronary artery bypass grafting (CABG) emergency operation in a COVID-19 patient, who presented unstable angina and coronary artery dissection during cardiac catheterization and percutaneous transluminal coronary angioplasty (PTCA). The Coronavirus disease 2019 (COVID-19) pandemic outbreak, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a health emergency worldwide.Several cases reported in literature showed, despite the most common lung impairment, a cardiovascular involvement, which reflects a complex interaction between the high capacity of Sars-CoV-2 replication and a series of manifestations, often leading to a poor prognosis. A pivotal role is played by the endothelium. Physiologically, the vascular endothelium is in charge of the maintenance of the homeostasis through a fine regulation of immune competence, inflammatory equilibrium, tight junctional barriers, hemodynamic tone, and stability as well as optimally balanced thrombotic and fibrinolytic pathways. When affected by the exacerbated inflammatory cascade triggered by Covid-19, a sequelae of events lead to an alteration of the vasomotor tone and a deregulation of clotting factors, finally exerting a key-role in the magnitude of Sars-CoV-2 infection severity.Therefore, pre-existing conditions that affect the cardiovascular system, either acquired or congenital, may explain different expressions of systemic impairment, as seen in African American descendant populations . BesidesWe present a report of a successful coronary artery bypass grafting (CABG) emergency operation in a COVID-19 patient, who presented unstable angina and coronary artery dissection during cardiac catheterization and percutaneous transluminal coronary angioplasty (PTCA).A 57 years-old female, tested positive for Sars-CoV-2 and in therapy with Azithromycin 500 mg and enoxaparin sodium 4000 UI daily, has been admitted to our hospital for chest pain.The anamnestic collection was positive for smoking, hypertension (HTN), dyslipidaemia, and hypothyroidism.At hospital admission laboratory tests showed high levels of LDH and high-sensitive cardiac troponin T (hs-cTnT), 257 UI/L and 0.156 μg/dL at initial assay, respectively, and 0.348 μg/dL at peak. No inflammatory markers elevations associated with COVID-19 were found .Non ST-elevated myocardial infarction (NSTEMI) was definitively diagnosed by electrocardiogram (EKG).Even though the patient did not show any symptoms suggestive for COVID-19, computed tomography (CT) of the chest detected bilateral ground-glass of the lung parenchyma, in absence of pleural or pericardial effusion .The patient, referred to cardiac catheterization laboratory, underwent coronary angiogram, which showed critical stenosis of both left anterior descending (LAD) coronary artery and first obtuse marginal (OM) coronary artery, and sub-occlusion of left circumflex (LCx) coronary artery.During intravascular ultrasound (IVUS) study, OM coronary artery was dissected and all the efforts accomplished in order to stabilize the anatomy with Percutaneous Coronary Intervention (PCI) failed due to retrograde progression of the dissection until both LCx and LAD coronary arteries .She underwent in emergency CABG surgery with cardiopulmonary bypass. She underwent routine aorta-right atrial cannulation for cardiopulmonary bypass. Myocardial protection with cold blood antegrade-retrograde cardioplegia was performed. Coronary revascularization was achieved using saphenous veins grafts [left internal mammary artery (LIMA) was not suitable because too small in size and a poor run-off] to LAD coronary artery and OM coronary artery. Both coronary vessels were extremely thin, dissected and with a very small residual lumen. Due to the hostile anatomy of the dissected arteries we disregarded the possibility of off-pump coronary revascularization. Surgery was uneventful with aortic cross-clamp and extracorporeal circulation times of 74 and 99 min, respectively.On post-operative day 1, the patient was extubated. Transfusions of 2 units of whole blood were used to manage post-operative anemia. Post-operative chest radiographies did not show atelectasis or pleural effusion. On day 4, the patient was admitted to the ward unit. She was treated with corticosteroids (dexamethasone 10 mg for the first 10 days and then tapered over by decreasing ¼ of the dose every 48 h), empirical antimicrobials  and prophylactic anticoagulant (enoxaparin sodium 4000 UI twice daily for the first 2 weeks and then 4000 UI one daily).Post-operative course was characterized by fever (T 38.8C°) on post-operative day 1 and 2 with a peak plasma interleukin-6 (IL-6) of 267 pg/mL  and by a progressive increase in D-dimer level from 665 to 4.015 μg/L.On post-operative day 21, she was discharged after a double negative nasopharyngeal swab test for Sars-CoV-2 in excellent general condition.We presented a case of ACS in a COVID-19 patient, complicated by coronary artery dissection during PTCA who underwent an emergency CABG surgery.Although clinical signs of severe sepsis were absent, CT findings were associated with COVID-19 pneumonia.Indeed, CT of the chest  showed tInterstitial lung disease increases the risks of post-operative pulmonary complications, such as acute lung injuries, acute respiratory distress syndromes (ARDS), pneumonias, atelectasis and pulmonary embolisms, among others. These significantly contribute to higher mortality rates, particularly in patients undergoing emergency surgeries .Confirming these findings, an international cohort study regarding 1,128 patients across 24 countries, who underwent surgery, showed that mortality and pulmonary complications were higher in patients with perioperative Sars-CoV-2 infection. Post-operative complications  occurred in half of patients due to inflammatory and/or immunosuppressive responses to surgery and mechanical ventilation .Therefore, despite the absence of evident symptomatology, our patient was exposed to a higher risk of post-operative pulmonary impairment.However, pre- and post-operative prophylactic treatment of our patient might have played a crucial role in reducing the incidence of these adverse outcomes.The patient was treated during isolation, before the onset of angina, with azithromycin 500 mg and prophylactic enoxaparin sodium 4000 UI one daily. Besides, in the post-operative period, she was treated with antimicrobial therapy, heparin, and corticosteroids.https://www.who.int/publications/i/item/WHO-2019-nCoV-Corticosteroids-2020.1) the use of systemic corticosteroids is strongly recommended for the management of severe and critical COVID-19, where the host immune response may drive the pathophysiology of the disease. Therefore, evidence suggested that the use of systemic corticosteroids reduced 28-day mortality in patients with critical and severe COVID-19  guidelines  . The bloSIRS may lead to an acute lung inflammation known as post-perfusion lung syndrome, in which TNF-α, IL-6, IL-8, and IL-1β have a pivotal role . Halter Similarly in severe COVID-19 emerging evidences suggest a correlation between increased IL-6 levels and the need for invasive mechanical ventilation . FurtherD-dimer specially in critical COVID-19 seems to be an emerging prognostic marker of hypercoagulability . In our Both CPB and Sars-CoV-2 infection trigger a “cytokine storm” which can synergistically lead to post-perfusion lung syndrome and ARDS . In the In order to clarify this aspect, cases of patients who underwent cardiac surgery and tested positive for Sars-CoV-2 RNA in the peri- operative period are reported.Rescigno et al.  presenteSimilarly, Fukuhara et al.  reportedOn the basis of these reports, we can hypothesize that undiagnosed COVID-19 infection might have precipitated inflammatory response after cardiac surgery.Yandrapalli et al.  reportedp < 0.001) until 48 h after surgery compared to controls and extracorporeal hemadsorption  for the publication of any potentially identifiable images or data included in this article.Each author has contributed significantly to this work. EG performed the conception and design of the study and final approval. SR performed the acquisition and interpretation of data and wrote manuscript. MV performed the interpretation of data and wrote the manuscript. MT, AL, and MP revised it critically for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ventricular septal rupture (VSR) is a rare complication of ST-elevation myocardial infarction (STEMI), typically discovered post-revascularization.We present the first case of VSR detected on point-of-care ultrasound (POCUS) in the emergency department immediately prior to emergent angiography, with management positively affected by this discovery. The VSR was quickly confirmed via right heart catheterization. Subsequently, hemodynamic stability was achieved using an intra-aortic balloon pump. A delayed surgical VSR repair, with concomitant coronary artery bypass grafting, was implemented for definitive management.This case highlights the utility of POCUS in a STEMI patient with a suspected mechanical complication. Ventricular septal rupture (VSR) is a serious complication of ST-elevation myocardial infarction (STEMI) associated with extremely high mortality. Development of VSR is thought to be mediated by the complete occlusion of a major vessel in the absence of spontaneous reperfusion or collateral circulation. In this regard, VSR is less likely to occur in a patient with a history of symptomatic coronary artery disease (CAD).2Diagnosis of VSR generally occurs within the first week post-MI, typically presenting as a clinical decompensation in patients who received timely revascularization. Whereas the median time from symptom onset to diagnosis is approximately five days, earlier occurrence (≤ two days) has been associated with thrombolytic utilization. In roughly 50% of cases, VSR occurs with total occlusion of the infarct-related artery. Anterior and inferior-posterior infarcts occur with roughly equal frequency. Hemodynamic instability often accompanies VSR. Cardiogenic shock is present in 39% of cases, and cardiac arrest occurs in 6.1% of cases.2) 89%. He required four liters per minute supplemental oxygen to maintain SpO2 greater than 94%. An electrocardiogram revealed Q waves and ST-segment elevations in leads II, III, and aVF with reciprocal depressions in leads I, aVL, and V2–V5 . His high sensitivity troponin T was 5853 nanograms (ng)/L .A 61-year-old man with a history of long-term cigarette smoking (45 pack-years) presented to the ED with acute dyspnea and one week of chest pain. Initial vital signs were as follows: temperature 36°C, heart rate 97 beats per minute, blood pressure 176/100 millimeters of mercury, respiratory rate 30 breaths per minute, and oxygen saturation  is a devastating ST-elevation myocardial infarction (STEMI) complication that requires prompt diagnosis and management.What makes this presentation of disease reportable?Early discovery of a post-STEMI VSR on point-of-care ultrasound (POCUS) allowed for optimal management and led to a positive outcome.What is the major learning point?Immediate echocardiographic assessment should be performed whenever post-STEMI mechanical complications are suspected.How might this improve emergency medicine practice?Rapid screening of the unstable STEMI patient using POCUS can help diagnose VSR and facilitate appropriate management.2)  and the pulmonary-to-systemic flow ratio (Qp:Qs) was 7.8 , consistent with an extremely large left-to-right intraventricular shunt. Oxygen saturation measurements collected during right heart catheterization (RHC) are shown in the table.On POCUS in the ED, parasternal long- , Panel BThe coronary artery stenotic lesions were not amenable to stenting. Hemodynamic stabilization was achieved with placement of an intra-aortic balloon pump (IABP). On hospital day 10, single vessel coronary artery bypass grafting (CABG) and VSR repair were completed successfully. The IABP was removed the following day. Post-op transthoracic echocardiography (TTE) demonstrated moderately reduced left ventricular ejection fraction  and trivial left-to-right shunting. His post-op course was uncomplicated, and the patient was discharged home on hospital day 15.At two-week post-discharge follow-up with his new cardiologist, the patient endorsed improving exertional fatigue and was otherwise asymptomatic. His recovery had been uneventful. He remained abstinent from cigarette smoking.p:Qs of 1.4, and cardiac index of 2.3 L/min/m2 , and a borderline increase in left-to-right shunting. RHC showed a mild step-up in oxygen saturation from the mixed venous to the right ventricle , with a calculated QL/min/m2 .Mechanical complications of STEMI are life-threatening and necessitate prompt diagnosis and management. Concerning findings  should raise suspicion and trigger immediate echocardiographic assessment.Correcting the VSR is essential, with surgical repair representing the gold standard approach. In medically treated patients, the prognosis is grave, with in-hospital mortality ranging from 94–100%.13The advent and wide availability of mechanical circulatory support (MCS) devices has dramatically altered VSR management. The hemodynamic stabilization afforded by MCS allows for recovery of end-organ injury and serves as a bridge to definitive repair. Current MCS options include IABP, left ventricular assist devices , and extracorporeal membrane oxygenation. Currently, European Society of Cardiology and American College of Cardiology Foundation/American Heart Association guidelines suggest using IABP as a stabilizing measure.13It is uncertain whether revascularization with concomitant CABG at the time of VSR repair improves outcomes. Some studies fail to demonstrate a benefit, instead finding an association between the number of anastomoses and worse mid- to long-term outcomes.VSR is a rare but devastating complication that typically occurs three to five days post-myocardial infarction and requires definitive surgical repair. When a post-STEMI VSR or other mechanical complication is suspected, immediate echocardiographic assessment should be performed. Our case highlights that this can be accomplished using POCUS. Immediate VSR repair carries a high mortality risk due to the fragility of necrotic tissue. When feasible, a delayed approach enables a more durable repair of scarred tissues, markedly improving outcomes. Performing concomitant CABG at the time of VSR repair is controversial but may improve long-term survival.VideoModified apical four-chamber view on point-of-care ultrasound. The ventricular septal rupture is directly visualized by sweeping the imaging plane through the interventricular septum. Color flow Doppler demonstrates left-to-right blood flow across the defect.


Mycobacterial culture remains the gold standard for the diagnosis of tuberculosis. However, an appropriate digestion and decontamination method (DDM) is essential for the effective recovery of tubercle bacilli in culture. Therefore, the current study was designed to compare the performance of papain-cetylpyridinium chloride [papain-CPC] and pepsin-cetylpyridinium chloride [pepsin-CPC] DDMs against N-acetyl L-Cysteine-sodium hydroxide (NALC-NaOH) DDM for recovery of mycobacteria from clinically suspected pulmonary tuberculosis cases. To evaluate papain-CPC, pepsin-CPC and NALC-NaOH DDMs, sputum samples (N = 1381) were cultured on Löwenstein-Jensen medium and the results were compared. The papain-CPC DDM showed sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 93.27%, 71.7%, and 100%, respectively as compared to NALC-NaOH DDM. Similarly, pepsin-CPC DDM demonstrated sensitivity, specificity, positive predictive value and negative predictive value of 98.94%, 94.7%, 76.11%, and 99.81%, respectively. In summary, both papain-CPC and pepsin-CPC DDMs are highly sensitive and specific techniques for recovery of mycobacteria as compared to NALC-NaOH DDM. However, when the overall performances of all DDMs compared, papain-CPC DDM isolated increased number of mycobacterial isolates with comparatively higher numbers of colonies on LJ media than both pepsin-CPC and NALC-NaOH DDMs, indicating its potential to replace the NALC-NaOH DDM for recovery of mycobacteria from sputum samples. Mycobacterium tuberculosis (MTB) in culture. In the past years, various DDMs have been established to isolate MTB from clinical specimens  and 4 g of papain  in 100 ml of distilled water] was added to a 14 ml BD Falcon tube containing 2–3 ml of sputum aliquot. The contents were mixed by vortexing (1 min), and were incubated at 37°C for 30 min. After incubation, the volume was brought to 14 ml with the addition of distilled water. Then, this mixture was thoroughly mixed by inversions and vortexing (1 min) and centrifuged at 3000 x g for 20 min . The supernatant was carefully decanted and the remaining sediment was re-suspended in 200 μl of distilled water. This re-suspended pellet (0.2 ml) was inoculated on two slants of LJ media (one media with glycerol and the other with sodium pyruvate) followed by incubation at 37°C  for 8 weeks aerobically with manual monitoring of once per week for the growth of MTB.For pepsin-CPC DDM, 2–3 ml of sputum aliquot was mixed with equal quantity (2–3 ml) of 4% pepsin-CPC working solution [the stock solution of pepsin and CPC were prepared by dissolving 4 g of pepsin  and 4 g of CPC  in 100 ml of distilled water] and vortexed (1 min). Then, pepsin-CPC method was performed as described in the papain-CPC DDM.5'-CCT GCG AGC GTA GGC GTC GG 3' and T5-5' CTC GTC CAG CGC CGC TTC GG 3'  and the expected band size was 123 bps . In the current study, all the MTB isolates , were further confirmed by IS6110 PCR assay. However, this assay couldn’t amplify DNA of five MTB isolates that were identified by conventional methods. This may be probably due to the absence of IS6110 elements in these MTB strains or may also be due to the presence of some PCR inhibitors present. The current study used sputum specimens that were collected at microscopy-designated center of RNTCP (for routine microscopic examination) to presumptively diagnose PTB. However, final diagnosis of PTB was based on direct sputum smear, culture, chest X-ray examination, histopathological evidences and clinical characteristics or as recommended by trained physician. Patients with smear positive cases (and other investigations suggestive of TB) usually started with treatment as per the RNTCP guidelines [The current study evaluated the performance of NALC-NaOH DDM (reference method) with newly proposed papain-CPC and pepsin-CPC DDMs for the recovery of mycobacteria from sputum specimens that collected from clinically suspected PTB patients. The digested and decontaminated specimens were inoculated on two slants of LJ media; one LJ media with sodium pyruvate  between all the DDMs studied  after treatment with NALC-NaOH DDM. This higher recovery rate of MTB in culture (as compared to direct LED FM) is likely due to the increased sensitivity of culture that detects 10–100 bacilli/ml of specimen. Further, NALC-NaOH DDM possesses few limitations such as; technically intricate, instability of NALC solution, and relatively expensive (5g cost ~ 500 INR). In the present study, a diminished colony count was also noted when samples were digested and decontaminated using NALC-NaOH DDM as compared to papain-CPC and pepsin-CPC DDMs. This reduction in number of colonies may be due to death of bacilli in the sputum samples because of the toxicity induced by NaOH during the treatment procedure. In this study, around 20% of the positive cultures were isolated on 8 studied . However4 colony-forming units per ml [In the papain-CPC and pepsin CPC DDMs, both papain and pepsin functions as mucolytic agent and CPC as decontaminating agent. In our earlier study, it was found that overnight incubation of sputum with 1% CPC could effectively decontaminate the specimens . Howevers per ml . The oths per ml . Howevers per ml . Althougs per ml , 28. NevIn summary, both papain-CPC and pepsin-CPC DDM could effectively isolate reasonably higher number of mycobacterial isolates particularly in sputum specimens with smear-negative cases as compared to NALC-NaOH DDM. However, when the overall performances of both papain-CPC and pepsin-CPC DDMs compared, papain-CPC DDM could isolate increased number of mycobacterial isolates with comparatively higher number of colonies on LJ media as compared to both pepsin-CPC DDM and NALC-NaOH DDM, indicating the potential of papain-CPC DDM to replace the currently available NALC-NaOH DDM for the recovery of mycobacteria from sputum samples.


RNA interference (RNAi) provides the means for alternative antiviral therapy. Delivery of RNAi in the form of short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA) have demonstrated efficacy in gene silencing for therapeutic applications against viral diseases. Bioinformatics has played an important role in the design of efficient RNAi sequences targeting various pathogenic viruses. However, stability and delivery of RNAi molecules have presented serious obstacles for reaching therapeutic efficacy. For this reason, RNA modifications and formulation of nanoparticles have proven useful for non-viral delivery of RNAi molecules. On the other hand, utilization of viral vectors and particularly self-replicating RNA virus vectors can be considered as an attractive alternative. In this review, examples of antiviral therapy applying RNAi-based approaches in various animal models will be described. Due to the current coronavirus pandemic, a special emphasis will be dedicated to targeting Coronavirus Disease-19 (COVID-19). Since idoxuridine, the first anti-herpesvirus antiviral drug, reached the market in 1963 more than one hundred antiviral drugs have been formally approved . In thisAlternative strategies for antiviral drugs are naturally addressed currently including 5-substituted 2′-deoxyuridine analogues, nucleoside and pyrophosphate analogues, reverse transcriptase inhibitors, protease and integrase inhibitors, entry inhibitors, acyclic guanosine and nucleoside phosphonate analogues, immunostimulators, interferons and antimitotic inhibitors . One appCaenorabditis elegans [RNA-based gene silencing was originally discovered in plants and  elegans . More re elegans ,10 and m elegans  with mor elegans ,13. The  elegans . In addi elegans . Deliver elegans . Gene si elegans . The mec elegans . This apIn comparison to small-molecule or antibody-based drugs, the major advantage of RNAi-based strategies relates to the specific targeting of the drug function to a specific nucleotide sequence . In thisBioinformatics plays an important role in supporting RNAi-based antiviral therapy. For instance, a stochastic computational model has been designed to aid the development of anti-HIV RNAi therapies . The modAs with all therapeutic approaches, delivery plays an essential role also for RNAi-based drugs, which has also been reviewed previously . In thisAn alternative delivery approach comprises the use of viral vectors. In this context, for instance adenovirus (Ad) , adeno-aCurrently, there are many viral pathogens, such as HCV, human papilloma virus (HPV), human immunodeficiency virus (HIV), Ebola virus (EBOV), Dengue virus (DENV), and Chikungunya virus (CHIKV) just to name a few, that can be considered a threat to global human health . SARS-CoAdenovirus (Ad) vectors have also been applied for RNAi-based gene silencing, targeting the mosquito-borne flavivirus Tembusu virus (TMUV) . Based oIn another approach, the target for antiviral drugs was Ad as immunocompromised patients infected with Ad can develop life-threatening conditions . In thisRetrovirus vectors have been considered as the prototype for gene therapy, and although providing therapeutic efficacy of X-linked inherited immunodeficiency, unfortunately chromosomal integration induced the activation of the proto-oncogene LIM domain only 2 (LMO2) . For thiRNAi-based gene silencing has been explored using a replicating HIV-based lentivirus vector for the delivery of an antiviral shRNA cassette into HIV-1 susceptible cells with the aim of blocking chronic HIV-1 infection . The engIn the context of HIV-1-based gene silencing, anti-HIV shRNA and miRNA approaches can be compromised by their potential targeting of HIV-derived sequences in the vector leading to severely impaired vector titers ,68. The In another strategy, multiplexed miRNA-based shRNAs have been applied for simultaneous suppression of multiple HIV genes . MultiplIn an application of self-replicating RNA viruses, the negative-strand single-stranded (ssRNA) oncolytic VSV was used for the expression of the Nodamura virus protein B2, a known inhibitor of RNAi-mediated immune responses . In thisAlthough not based on viral vectors, it is appropriate to provide a summary of RNAi-based antiviral drug development utilizing non-viral delivery  with a sIn the case of flaviviruses, the global threat of Dengue virus (DENV) infections has accelerated the development of antiviral drug development, which has included several studies on siRNAs applied for the inhibition of DENV replication . For insThe current COVID-19 pandemic has made coronaviruses the most urgent targets for development of antiviral drugs, including the exploration of RNAi approaches . NaturalIn the context of MERS-CoV, four miRNAs and five siRNAs targeting the ORF1ab region have been rationally designed by computational calculations for gene silencing . RNAi haIn summary, RNAi-based gene silencing has presented great promise on several levels. Basic research has gained immensely as RNAi has together with proteomics become the most dynamic field of biotechnology . RNAi haThe current COVID-19 pandemic has also demanded the exploration of all possible alternatives for the discovery of novel or repurposed antiviral drugs for SARS-CoV-2 including RNAi. Finally, it is appropriate to briefly summarize the status of RNAi-based drugs, although they represent other disease indications than viral infections. In this context, RNAi-based treatment of neovascular age-related macular degeneration (Sirna-027) showed a good safety profile in 26 patients in a phase I study , but the


The phase changes within the thin films are studied by a combined approach using X-ray diffraction and transmission electron microscopy. The results reveal different phase transitions such as crystalline-to-amorphous phase changes, interface assisted crystallization of the cubic GST phase and structural transformations within crystalline phases. In particular, it is found that crystalline interfaces serve as crystallization templates for epitaxial formation of metastable cubic GST phase upon phase transitions. By varying the laser fluence, GST thin films consisting of multiple phases and different amorphous to crystalline volume ratios can be achieved in this approach, offering a possibility of multilevel data storage and realization of memory devices with very low resistance drift. In addition, this work demonstrates amorphization and crystallization of GST thin films by using only one UV laser with one single pulse duration and one wavelength. Overall, the presented results offer new perspectives on switching pathways in Ge-Sb-Te-based materials and show the potential of epitaxial Ge-Sb-Te thin films for applications in advanced phase change memory concepts.Ge-Sb-Te-based phase change memory alloys have recently attracted a lot of attention due to their promising applications in the fields of photonics, non-volatile data storage, and neuromorphic computing. Of particular interest is the understanding of the structural changes and underlying mechanisms induced by short optical pulses. This work reports on structural changes induced by single nanosecond UV laser pulses in amorphous and epitaxial Ge The working principle of conventional phase change memory is based on fast reversible phase changes between crystalline and amorphous phases of Ge-Sb-Te materials ,2,3. For2Sb2Te5 (GST) thin films induced by single UV ns-laser pulse in order to explore possible switching pathways, which might represent optimized transitions in terms of entropic loss, switching times, and energy consumption.Thus, this work aims to study structural transitions in amorphous and layered epitaxial Ge−8 mbar and a constant Ar gas flow was introduced to moderate the kinetic energy of the ablated species. The as-grown films were finally capped with an optically transparent LaAlO3-x (LAO) protective layer of about 5 nm thickness to protect the GST thin films against oxidation. The structure of as-grown and laser irradiated thin films was studied ex-situ by X-ray diffraction (XRD) measurements . In order to induce structural transitions, the GST thin films were irradiated with a single laser pulse at a wavelength of 248 nm and pulse duration of 20 ns. For the optical switching experiments, the same laser was used as for the PLD growth of GST thin films. The temporal pulse shape was Top Hat. The size of the laser beam on the surface of the thin films was 0.5 cm × 1 cm. The area of X-ray probing was smaller than the area of GST thin films irradiated by the UV laser.Amorphous and epitaxial GST thin films were deposited on Si (111) substrates by pulsed laser deposition (PLD) . A KrF e3 G2 60–300 transmission electron microscope operating at 300 kV accelerating voltage. Bright-field transmission electron microscopy (BF-TEM) and high-resolution transmission electron microscopy (HRTEM) images as well as selected area electron diffraction (SAED) patterns were acquired using a Gatan CCD camera . For high-resolution scanning TEM (HRSTEM), a probe forming annular aperture of 25 mrad was used and all images were recorded with a high-angle annular dark-field (HAADF) detector using annular ranges of 80–200 mrad. The preparation of TEM specimens was performed by a combination of focused high- and low-energy ion beam milling. The microstructure of GST thin films was investigated in a probe Cs-corrected TitanFirst, crystalline-to-amorphous phase transitions induced by single ns-laser pulse irradiation of epitaxial GST thin films with vacancy ordered metastable phase (vo-GST) were studied. 2, the epitaxial vo-GST thin film undergoes a phase transition to an amorphous GST (a-GST) thin film, which becomes apparent by the corresponding ω−2θ pattern showing only substrate contributions  substrate. Here, the GST grains are in direct contact with the substrate. This is a strong evidence for a laser induced epitaxial crystallization starting at the substrate surface. Thus, in this case, the relation between laser fluence and the thin film thickness leads to a sufficient crystallization temperature at the Si/a-GST interface, where an epitaxial crystallization process starts at the substrate surface and propagates towards the thin film surface. It should be noted that epitaxial recrystallization of GST thin films grown by molecular beam epitaxy on Si (111) substrates can be also achieved by using femtosecond laser pulses [lections b. In conr pulses .The results in 2 fluence in order to incrementally increase the crystalline fraction of the thin film. After each applied laser pulse, a XRD pattern was recorded and these are depicted in The next step of this study evaluates whether the a-GST/c-GST interface can be forced to propagate epitaxially towards the surface of the thin film by the accumulation of multiple laser pulses. That is, whether c-GST also forms epitaxially at an already existing a-GST/c-GST boundary. For this purpose, a further laser beam irradiation experiment is conceived. In detail, an amorphous GST thin film is deposited on a crystalline Si (111) substrate and crystallized by successive single ns-laser pulses at 21 mJ/cmThus far, conventional crystalline-to-amorphous phase transitions have been studied by applying intense laser pulses to the as-deposited epitaxial GST thin films in order to initiate a melt quenching process leading to amorphization. Epitaxial GST thin films could, however, offer the opportunity to follow even small structural changes without initiating a complete amorphization. Therefore, epitaxial GST thin films were irradiated by ns-laser pulses at moderate laser fluences in order to assess whether even minor structural modifications can be triggered and, for example, whether a small thermal influx could lead to a randomization of the Ge/Sb/Vacancy sublattice in a vo-GST thin film.2. Remarkably, despite the comparatively low laser fluence applied, significant changes in the XRD pattern are observed. In particular, a drastic reduction of the superstructure reflections attributed to the periodically spaced VLs becomes apparent. However, two pronounced peaks associated with c-GST (111) and c-GST (222) reflections remained. This indicates that the ns-laser irradiation not necessarily causes an amorphization process but rather a transition from one crystalline phase to another. In detail, the XRD results suggest an intracrystalline phase transition from vo-GST to c-GST.2. The film can be divided into three distinct regions . According to the image contrast and corresponding fast Fourier transformation (FFT) patterns, the regions c-GST and t-GST represent cubic and trigonal crystalline structures, respectively. The FFT pattern calculated from region t-GST in In order to clarify the phase transition observed above , furtherThe region a-GST in Ultimately, the above-presented results show a rapid transition from epitaxial vo-GST or t-GST thin films to epitaxial c-GST thin films. In this part of the study, the observed process is first subjected to a full switching cycle. It is known that the reverse process, i.e., the formation of vo-GST can be achieved by thermal heating ,29,30,32In conclusion, structural transitions in amorphous and layered epitaxial GST thin films (t-GST and vo-GST) induced by a single UV ns-laser pulse were studied by a combined approach using XRD and TEM analysis. Particularly, this work demonstrates different structural transformations, such as crystalline-to-amorphous phase changes, interface-assisted epitaxial crystallization, and phase transformations within crystalline phases. The amorphization of epitaxial vo-GST thin films and their recrystallization to c-GST thin films were achieved by applying optical pulses of high and low laser fluences, respectively. However, laser pulses of moderate fluences resulted in a single step structural transition to the c-GST phase. The phase was formed from a transient molten phase at the melt-crystalline interface upon the cooling process, providing access for identification of crystal growth velocities in epitaxial GST thin films . It was


Thoracic cancers, including lung cancer, esophageal cancer, and mesothelioma, accounted for nearly 250,000 new diagnoses in 2018 . TherefoResistance to apoptosis is a well-established hallmark of cancer . Mitochoin vitro malignant transformation model to develop a systematic approach to test whether Minority MOMP was involved in bile acid-induced esophageal carcinogenesis [In our recent study published in Oncogene, we established an ogenesis . In our ogenesis . These fNext, we queried whether Minority MOMP was associated with resistance to apoptosis. Given that the apoptotic machinery was activated, did this mechanism simultaneously enable resistance to prevent Minority MOMP from shifting to complete MOMP? We explored whether the known Bcl-2 proteins that are associated with resistance to apoptosis were upregulated. We found that Mcl-1 expression was significantly increased in DCA-T cells. Therefore, we knocked down Mcl-1 and noted decreased mitochondrial membrane potential, increased cytoC release, and an increase of caspase activation beyond the threshold in which the cells could survive. These results indicate that Mcl-1 is critical for maintenance of Minority MOMP and blocking MCL-1 could shift Minority MOMP to complete MOMP.Mcl-1 acts as a switch that can convert Minority MOMP to complete MOMP. Therefore, targeting Mcl-1 may be a therapeutic strategy in combination with other anti-cancer drugs. Mcl-1 inhibition is known to be effective against several solid, cancer-derived cell lines . To evalIn summary, Minority MOMP is a newly discovered mechanism that may be common among cancers induced by environmental carcinogens. While Minority MOMP promotes carcinogenesis, it may simultaneously upregulate anti-apoptotic proteins to protect the cancer cell from undergoing apoptosis. By identifying and directly targeting these proteins, the reestablishment of cellular sensitivity to apoptosis through the disruption of Minority MOMP may overcome resistance in treatment-refractory cancers . Additio


One striking feature of the large KRAB domain-containing zinc finger protein (KZFP) family is its rapid evolution, leading to hundreds of member genes with various origination time in a certain mammalian genome. However, a comprehensive genome-wide and across-taxa analysis of the structural and expressional features of KZFPs with different origination time is lacking. This type of analysis will provide valuable clues about the functional characteristics of this special family.In this study, we found several conserved paradoxical phenomena about this issue. 1) Ordinary young domains/proteins tend to be disordered, but most of KRAB domains are completely structured in 64 representative species across the superclass of Sarcopterygii and most of KZFPs are also highly structured, indicating their rigid and unique structural and functional characteristics; as exceptions, old-zinc-finger-containing KZFPs have relatively disordered KRAB domains and linker regions, contributing to diverse interacting partners and functions. 2) In general, young or highly structured proteins tend to be spatiotemporal specific and have low abundance. However, by integrated analysis of 29 RNA-seq datasets, including 725 samples across early embryonic development, embryonic stem cell differentiation, embryonic and adult organs, tissues in 7 mammals, we found that KZFPs tend to express ubiquitously with medium abundance regardless of evolutionary age and structural disorder degree, indicating the wide functional requirements of KZFPs in various states. 3) Clustering and correlation analysis reveal that there are differential expression patterns across different spatiotemporal states, suggesting the specific-high-expression KZFPs may play important roles in the corresponding states. In particular, part of young-zinc-finger-containing KZFPs are highly expressed in early embryonic development and ESCs differentiation into endoderm or mesoderm. Co-expression analysis revealed that young-zinc-finger-containing KZFPs are significantly enriched in five co-expression modules. Among them, one module, including 13 young-zinc-finger-containing KZFPs, showed an ‘early-high and late-low’ expression pattern. Further functional analysis revealed that they may function in early embryonic development and ESC differentiation via participating in cell cycle related processes.This study shows the conserved and special structural, expressional features of KZFPs, providing new clues about their functional characteristics and potential causes of their rapid evolution.The online version contains supplementary material available at 10.1186/s12860-021-00346-w. KRAB domain-containing zinc finger protein (KZFP) family is the largest family of transcription factors in mammals . For exaAs the result of the rapid evolution of this family, KZFP genes with various evolutionary ages exist in the current genomes , 5, 7. TStructural and expressional features are closely related to the functional characteristics of proteins. The protein intrinsic disorder degree, one of the key structural features, affects protein function and protein-protein interaction network , 11, andIn this study, we systematically explored the relationships between evolutionary age and structural disorder features of KZFPs, the expression width or expression level across early embryonic development, ESCs differentiation, embryonic and adult organs, tissues in 7 mammals. In total, 29 RNA-seq datasets, including 725 samples were involved in the analysis. Some conserved paradoxical phenomena were observed in these analyses, providing new clues about their functional characteristics and the potential causes of the rapid evolution of this large family.For the protein domains in chordates, those originating after the origination of the common ancestor of chordates are usually regarded as evolutionarily young domains , 21. In Homo sapiens), chimpanzee (Pan troglodytes), rhesus (Macaca mulatta), mouse (Mus musculus), rat (Rattus norvegicus), cattle (Bos taurus) and opossum (Monodelphis domestica), we found that there are no significant differences between KZFPs with different gene age grades . Similarly, we found that there were no significant differences in disorder degree of those regions in KZFPs among different gene age grades  and the expression width of each gene was calculated. The samples included the embryos of early development, different time points during ESCs differentiation into three germ layers and the subsequent terminal-differentiated cells, embryonic development of various organs and various adult tissues or organs. The number of samples in which a certain gene expressed was defined as the expression width of the gene.It is obvious that young genes  tend to be expressed at specific timepoints or spaces, and old genes tend to be wildly expressed Fig. a. To oure. g., KZFPs). For PCGs, genes encoding completely structured proteins tend to be expressed spatiotemporal specifically compared with disordered proteins  and gene expression abundance. As the results, gene age is positively correlated with gene expression abundance for PCGs in almost all samples Fig. . HoweverWe further used hierarchical clustering method to analyze the normalized expression data of KZFPs in different samples, and found that the same or similar samples in different data sets could be preferentially clustered together   was perfIn total, 134 young-zinc-finger-containing KZFPs were involved in 17 out of the 23 EM modules, among which 8 modules contain more young-zinc-finger-containing KZFPs than the overall ratio. The representative enriched GO terms  for each module are shown for these 8 EMs Fig. a. These In order to obtain the details about co-expression of genes in EM7, we calculated weighted topological overlap (wTO)  to obtaiWe also analyzed the interactors of these young-zinc-finger-containing KZFPs in EM7. Since the protein-protein interaction data we used was based on HEK293 cells  and HEK2Among the 18 BMs, we found that BM5, with a high proportion of old-zinc-finger-containing KZFPs, tend to be involved in the functions closely related to brain development, such as nervous system development  in all gene age grade are highly structured; as exceptions, old-zinc-finger-containing KZFPs  have relatively disordered KRAB domains and linker regions. The conformation of highly structured proteins and domains usually are rigid , which aSecondly, young genes, especially those encoding highly structured proteins, are generally expressed with a spatiotemporal-specific pattern –23, howee. g. ZNF267) are specifically recruited into embryonic development and the differentiation from ESCs to endoderm or mesoderm, such as ZFP809 silencing endogenous retroelements in ESCs and embryonic development [e. g. ZNF764) tend to participate in functions related to brain development. These KZFPs may also inhibit TEs by interacting with KAP1 in brain [Furthermore, KZFPs containing young zinc fingers are preferentially recruited into functions related to early development-related processes, suggesting that young-zinc-finger-containing KZFPs . Evolutionary distance between species were estimated by TimeTree [The protein sequences of 64 species from 64 genera across the superclass of Sarcopterygii ); chimpanzee adult organs (GSE69241); rhesus early embryonic development (GSE103313); organs at developmental stages from early organogenesis to adulthood in rhesus (E-MTAB-6813); rhesus adult organs (GSE69241); mouse early embryonic development ; differentiation of mESCs into endoderm (GSE92572); differentiation of mESCs into mesoderm (GSE47948); differentiation of mESCs into ectoderm (GSE94364); organs at developmental stages from early organogenesis to adulthood in mouse (E-MTAB-6798); mouse adult tissues/organs ; organs at developmental stages from early organogenesis to adulthood in rat (E-MTAB-6811); cattle early embryonic development (GSE143848); cattle adult organs (GSE41637); organs at developmental stages from early organogenesis to adulthood in opossum (E-MTAB-6833).The RNA-Seq data was downloaded from GEO and ArrayExpress database: human early embryonic development ; differentiation of hESCs into endoderm ; differentiation of hESCs into mesoderm ; differentiation of hESCs into ectoderm ; organs at developmental stages from early organogenesis to adulthood in human (E-MTAB-6814); human adult tissues/organs   at developmental stages from early organogenesis to adulthood in human as a dataset related to the middle and late development stages. For each dataset, TPM values were loge sample   or log (p).This was performed using the method as previously described . BrieflyFor GO term enrichment analysis, we first removed genes without GO term annotations. Subsequently, we retained the genes expressed in at least one sample as the background.Additional file 1: Figure S1. The schematic diagram of the domain architecture of KZFP and the key amino acids in zinc finger binding to DNA. Figure S2. The SDR values of KRAB domains with different gene age grades in 7 mammals. Figure S3. The expression pattern of KZFP genes with different zinc finger divergence time grades. Figure S4. The expression pattern of KZFP genes in 7 mammals. Figure S5. The highly expressed KZFP genes in human. Figure S6. The expressional and functional characteristics of the co-expression modules containing old-zinc-finger-containing KZFPs. Figure S7. The PPIs of young- or old-zinc-finger-containing KZFPs. Figure S8. The SDR values and interactors of ZKSCAN3, ZNF287 and ZNF90. Figure S9. The WGCNA parameter for early development dataset (A) and brain development dataset (B).Additional file 2. Percentage of each disorder grade of KRAB domains and other chordata-specific domains in each of the 64 species  and expression level (TPM) in 7 mammals (related to Fig. Additional file 7. The expression z-score of KZFPs in 7 representative mammals (related to Figure S4 & S5 in Additional file Additional file 8. The wTO of genes in EM7 or BM5 (related to Fig. Additional file 9. The protein-protein interaction network of human KZFPs and the expression of baits and preys in each sample.Additional file 10. The number of PCGs and KZFP genes in each evolutionary age grade in 7 mammals.