While antibodies can provide significant protection from SARS-CoV-2 infection and disease sequelae, the specific attributes of the humoral response that contribute to immunity are incompletely defined.We employ machine learning to relate characteristics of the polyclonal antibody response raised by natural infection to diverse antibody effector functions and neutralization potency with the goal of generating both accurate predictions of each activity based on antibody response profiles as well as insights into antibody mechanisms of action.To this end, antibody-mediated phagocytosis, cytotoxicity, complement deposition, and neutralization were accurately predicted from biophysical antibody profiles in both discovery and validation cohorts. These models identified SARS-CoV-2-specific IgM as a key predictor of neutralization activity whose mechanistic relevance was supported experimentally by depletion.Validated models of how different aspects of the humoral response relate to antiviral antibody activities suggest desirable attributes to recapitulate by vaccination or other antibody-based interventions.The online version contains supplementary material available at 10.1186/s12865-022-00480-w. The SARS-CoV-2 pandemic has resulted in over 300 million cases, 5.5 million deaths, and unprecedented social, economic, and educational impact despite interventions that have included quarantines, shutdowns, social distancing, and masking requirements. However, the pandemic has also led to international collaborations working toward understanding the disease and developing novel therapeutics and vaccines. To date, these efforts have resulted in several novel therapies and several vaccines approved for widespread deployment under emergency use authorization .The success of these vaccines is thought to result in no small part to the potent antiviral activities of the antibodies they induce. While reinfections have been documented , 3, seroHowever, whether elicited by vaccination or infection, antibody responses between individuals are highly variable –29, bothAntibody functions, including neutralization assessed by either an authentic virus assay or a luciferase-based pseudovirus assay, antibody-dependent cell-mediated phagocytosis (ADCP) mediated by monocytes, deposition of the complement cascade component C3b (ADCD), and FcγRIIIa ligation as a proxy for NK cell mediated antibody dependent cellular cytotoxicity (ADCC) induced by antibodies in response to recombinant antigen were previously reported  for a seTo understand how the different facets of the Ab response relate to one another, hierarchical clustering was performed on the biophysical antibody profiles of convalescent plasma donors (JHMI) and compared to the serum profiles of SARS-CoV-2 naïve subjects. Extensive variability in the SARS-CoV-2-specific Ab response magnitude and character was noted Fig. A. High lA weighted network plot depicting Pearson’s correlation coefficients between Fc array features and functional measurements was created to elucidate correlative relationships more directly between aspects of humoral responses Fig. B. As wasWith the dual goals of better understanding the humoral response features that may drive complex antibody functions and enabling robust predictions from surrogate measures, we applied supervised machine learning methods to this (JHMI) dataset, while using the DHMC cohort as validation to determine whether the models could predict activity in a generalized manner. A regularized generalized linear modeling approach trained to utilize Fc Array features to predict each antibody function with minimal mean squared error was selected based on prior success in identifying interpretable factors that contribute to functional activity while avoiding overfitting . Five-foThe model consistently selected a subset of features for each function Fig. A. The feTo evaluate the magnitudes of feature contributions, a representative model for each function demonstrating the identity and relative coefficients of the contributing features is presented Fig. B. Again,Beyond specificity, distinct antibody Fc characteristics contributed to model predictions. The most frequent Fc characteristic of features contributing to the final model of neutralization potency was the magnitude of IgG response, consistent with neutralization being FcR-independent. In contrast, the most frequent Fc characteristics in modeling ADCC and ADCP were FcγRIII- and FcγRII-binding responses, respectively—the receptors most relevant to each function. Further, despite comprising a relatively small fraction of circulating IgG, but consistent with its enhanced ability to drive effector functions , 50, IgGIntriguingly, S1-specific IgM contributed to models of neutralization potency. IgM is typically associated with initial exposures , suggestOverall, while functions were predicted with differing degrees of accuracy, each generalized well to the independent validation cohort and relied upon features with established biological relevance.Given the somewhat surprising appearance of an IgM feature in predictions of neutralization activity, we sought to evaluate the mechanistic relevance of this isotype in particular. In a select group of individuals with both high IgM and neutralization levels n = 11), IgM was depleted from serum to determine whether the loss of CoV-2-specific IgM resulted in a reduction in neutralization of SARS-CoV-2  and has been previously described . The valHuman subject research was approved by both the Johns Hopkins University School of Medicine’s Institutional Review Board and the Dartmouth-Hitchcock Medical Center Committee for the Protection of Human Subjects. All participants provided written informed consent.The magnitude, Fv specificity, and Fc domain characteristics of antibody responses to diverse coronavirus and control antigens were profiled by multiplexed Fc Array assay , as prevAntibody functions were assayed as previously described , 45. BriIgM depletion.IgM was depleted from serum as described previously . BrieflyBasic analysis and visualization were performed using GraphPad Prism. Heatmaps, correlation plots, and other graphs were generated in R  was tuned using five-fold cross-validation to minimize mean squared error (MSE). A process of 200-times repeated modeling was used to investigate the potential of the different combinations of the biophysical features for modeling. Established with the JHMI cohort, a final model was selected based on the median MSE obtained among the repeated run in the JHMI cohort. The selected features and their coefficients were reported at a value of λ at which median model performance fell one standard error above the minimum to optimize the generalizability and provide more regularization to the model. In the permutation test procedure, the penalized multivariate regression was performed against randomized functional outcomes in the JHMI cohort in a 200-time repeated fashion. The correlation network was conducted with the biophysical features that were repeatedly selected within the repeated modeling process.Multivariate linear regression was employed to predict antibody functions based on biophysical features with the R package “Glmnet” , as prevAdditional file 1: Table S1. The expression of 1879 IRGs in GSE98793 dataset.


Genome-editing technologies have the potential to correct most genetic defects involved in blood disorders. In contrast to mutation-specific editing, targeted gene insertion can correct most of the mutations affecting the same gene with a single therapeutic strategy (gene replacement) or provide novel functions to edited cells (gene addition). Targeting a selected genomic harbor can reduce insertional mutagenesis risk, while enabling the exploitation of endogenous promoters, or selected chromatin contexts, to achieve specific transgene expression levels/patterns and the modulation of disease-modifier genes. In this review, we will discuss targeted gene insertion and the advantages and limitations of different genomic harbors currently under investigation for various gene therapy applications. Blood genetic disorders are caused by mutations in genes or in their regulatory elements that result in a dysfunctional, dysregulated, or absent protein. Conventional gene therapy approach consists of the addition of a functional copy of a mutated gene to patients' cells using viral vectors, such as adeno-associated virus (AAV) , limiting the choice of regulatory elements to include in the expression cassette, especially when delivering large transgenes  . AAV has a tendency to integrate at pre-existing chromosomal breaks that provide free DNA ends for non-homologous end joining (NHEJ)  endogenous promoters, when promoterless transgenes are inserted under the control of endogenous enhancers/promoters; (ii) safe genomic harbors, when transgenes and their promoters are integrated into genomic regions that allow robust expression without affecting cell physiology; (iii) disease modifier genes, when transgenes integrate into coding sequence of endogenous genes, whose inactivation benefits disease-affected cells; and (iv) specificity exchange, when transgenes are integrated into coding sequence of endogenous genes to change their function.It is worth noting that this subdivision is only a working framework, as the same integration site can fall into two or more categories, and it is not exhaustive, as new integration strategies are described every day.A straightforward approach for targeted gene replacement consists in inserting a functional copy of a gene downstream of its endogenous promoter. This strategy can correct most pathological mutations that are scattered along the gene body (such as substitutions and frameshift mutations), while maintaining physiological gene expression , which cex vivo to replace interleukin-2 receptor subunit gamma (IL2RG), whose mutational inactivation causes X-linked severe combined immunodeficiency (X-SCID) , the life-long source of all the different blood progenitors. Genovese via ZFN  cDNA cassette with AAV8 to the liver of a humanized mouse model of hemophilia B  deficiency, an AAV carrying a liver-specific promoter and a human OTC transgene was integrated via CRISPR/Cas9 in the murine OTC locus .Although gene-editing technologies are evolving at a fast pace, it can be challenging to correct enough cells to reach a clinical benefit even using high doses of nuclease and donor DNA, which increase chances of off-target genomic cleavage, immune responses, and donor random integration. An alternative strategy consists in “hijacking” strong endogenous promoters to overexpress therapeutic cassettes from few modified cells . An elegAlthough promising, this approach still presents some concerns. First, targeted integration can lower serum albumin levels  was historically identified as the preferential integration site of wild-type AAV in human cell lines  encodes for the main HIV co-receptor. Since a bi-allelic null mutation of this receptor (CCR5Δ32) confers HIV-1 resistance and is not associated with any major pathology  was serendipitously discovered in mice as a reliable site to integrate DNA cassettes for transgenesis  and, when β-globin chains are absent or limiting, free α-globin precipitates causing hemolysis and ineffective erythropoiesis. Reduction of α-globin has been shown to ameliorate the β-thalassemia phenotype   of interest under the control of the heavy or light immunoglobulin chain promoter to reprogram B cells to secrete broadly neutralizing Ab against pathogens, for which no protective Ab has been isolated  and episomal vector loss (AAV). The advent of the first generation of DNA endonucleases allowed the integration of transgenes in few selected genomic loci, mainly to achieve stable expression while minimizing insertional mutagenesis risk. Now, thanks to easily programmable nucleases such as CRISPR/Cas9, we have dramatically expanded our integration options and can creatively exploit different genomic locations to finely tune transgene expression or modulate disease-modifier genes to improve gene therapy outcomes.A common strategy to target transgene integration combines nucleases with a donor DNA template  and leverages the homologous recombination pathway. However, before clinical translation, strict functional validation will be necessary to reduce potential adverse events associated with each individual component of this system. In particular, nucleases can induce potential off-targets (Kleinstiver et al., Additional studies and further optimization of existing editing technologies will remove these hurdles and allow a broad clinical application of the described strategies to treat both monogenic and multifactorial blood diseases.GP and MA wrote the manuscript. 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.


Pearl powder is a well-known traditional Chinese medicine for a variety of indications from beauty care to healthcare. While used for over a thousand years, there has yet to be an in-depth understanding and review in this area. The use of pearl powder is particularly growing in the biomedical area with various benefits reported due to the active ingredients within the pearl matrix itself. In this review, we focus on the emerging biomedical applications of pearl powder, touching on applications of pearl powder in wound healing, bone repairing, treatment of skin conditions, and other health indications. Pearl powder has been used for more than a thousand years for traditional Chinese medicine, cosmetics, and as a health food substitute ,2. It is3) and magnesium carbonate are the primary ingredients of mollusc shells, with the remaining organic matrix containing proteins, glycoproteins, and polysaccharides [3(PO4)2), aluminium oxide, and iron oxide. Pearl also contains trace elements like sodium, manganese, selenium, aluminium, and copper. Animal studies have demonstrated that pearl powder has beneficial pharmacological effects such as anti-oxidant, anti-inflammatory (due to the magnesium that it possesses), anti-aging (through the stimulation of fibroblasts), immunomodulating, and wound healing [3 in the aragonite isomorph. Bones, on the other hand, consist mainly of Ca3(PO4)2 and hydroxyapatite (HA). Both exist naturally as composites via biomineralization and have a structural supporting function [Calcium carbonate , alumi healing ,13,14,15 healing , further healing . The irifunction ,19. PearThis review focuses on recent advances made in the use of pearl powder for biomedical applications, including wound healing, tissue engineering, and bone regeneration. Of note, we wish to highlight that the biochemistry and application of pearl powder are relatively nascent interests that are set to grow. The number of research papers in the study of biomineralization of pearls has risen steadily over the last 10 years . It is o3), one of the most abundant minerals on earth, is the main component in pearls, i.e., the nacre of mollusc shells and otoliths. Anhydrous CaCO3 can exist in three polymorphs depending on environmental conditions: calcite, aragonite, and vaterite, of which calcite is the most thermodynamically stable, followed by aragonite and vaterite [Calcium carbonate , matrix proteins and other soft organic biopolymers (ca 5 wt.%). Crystallization of CaCO3 is primarily stimulated by the organic matrix in the nacreous layer. Various organic matrices had been extracted and purified from the nacreous layer of mollusc shells using ethylenediaminetetraacetic acid (EDTA) or acetic acid, and were subsequently tested for in-vitro CaCO3 crystallization to clarify the mechanism of biomineralization in nacre [3 crystals. Gong et al. have identified a nacre matrix protein originated from the mantle epithelia of Pinctada fucata pearl that initiates crystal nucleation and calcium deposition with inconsistent morphology [P. fucata that contributes significantly in controlling the crystallization of CaCO3 and inducing the formation of needle-like aragonite crystals [Most freshwater pearls have lustre. Aragonite is the main inorganic component that contributes to the lustrous property of pearls, giving rise to the term “aragonite pearls” ,28. Vatein nacre ,31,32,33rphology . Zhang ecrystals . Most of2EDTA) is the primary extraction method of nacre organic matrix, but this method potentially undermines the structures or roles of proteins by sequestering essential metal ions. To overcome this, weak acid can be used to extract organic matrices from pearls [3 [3 solutions gradually and continually. To characterize the crystals, scanning electron microscopy (SEM) is utilized to observe the morphologies and Raman spectroscopy has also been proven to be a powerful technique to distinguish the polymorphs of CaCO3. Progress has been made using infrared reflectance spectroscopy as well. Mixtures of pearl powder and shell powder were calcined at various temperatures for different periods followed by analysis by infrared reflectance spectroscopy [−1 represents better consistency of pearls, while intense peaks at 1082 cm−1, 712 cm−1, and 699 cm−1 showed the opposite. Bands in ranges 1550–1350 cm−1, 730–680 cm−1, 830–880 cm−1, and 690–725 cm−1 were also useful for distinguishing pearl powders and recognizing adulteration.Decalcification by disodium ethylenediaminetetraacetate dihydrate (Nam pearls . Aizenbeearls [3 . This metroscopy . When catroscopy . Apart fPinctada maxima) was implanted into rat dermis and demonstrated that it fostered better skin tone while stimulating proper physiological functioning in skin fibroblast [Acute injuries, including burns and ulcers, are complex lesions that can be difficult to treat. Wound healing occurs in three phases, viz inflammation, proliferation, and maturation . Inflammbroblast , having broblast . Medium-Pinctada margaritifera and tested its effect on artificially dehydrated skin explants as a representation for atopic dermatitis [Hyriopsis cumingii) could induce oral fibroblast proliferation and collagen aggregation, suppress the activity of matrix metalloproteinase-2, and significantly promote TIMP-1 synthesis, benefiting wound healing [To further understand the role of active components of nacre on skin therapies, Rousseau et al. isolated lipids from the nacre of rmatitis . Nacre lrmatitis . In the rmatitis . The app healing . DiffereNanotechnology holds promise for revolutionizing approaches for tackling issues by unravelling the possibilities at a nanoscale level. Chen et al. have investigated pearl powders of various particle sizes for wound care using the rat skin excision model . Both miThe advancement of antimicrobial nanofibre dressings that provide protection to the injured tissues from commensal pathogens while promoting tissue regeneration at the same time would be significant in plastic and reconstructive surgery practices. Recently, Mayandi et al. studied the effect of chondroitin sulphate on the physicochemical properties of polydopamine crosslinked electrospun gelatin nanofibres containing mineralized magnesium . ComposiMytilus edulis and the Pacific oyster Crassostrea gigas) [Yang et al. used a room-temperature super-extraction method to generate the key active components of pearl and mixed the pearl extracts with poly(gamma-glutamic acid) hydrogels. The hydrogels with pearl extracts were effective in enhancing anti-inflammatory and anti-apoptotic function in human keratinocyte (HaCaT) cells exposed to low-dose ultraviolet B (UVB) , suggesta gigas) . Both exCaenorhabditis elegans due to its antioxidant activity. Both in vivo and in vitro studies indicated that pearl powder is an effective antioxidant and could potentially be used to treat multiple age-related degenerative diseases. Shao et al. showed that pearl powder is effective in beauty treatment and resistant to aging. However, particle size may influence antioxidant activity. Ultra-nano and ultra-micro pearl powders showed better free radical scavenging effects than water-soluble pearl powder [Lipid peroxidation can be induced by free radical oxidation and cause irreversible impairment of cellular macromolecules such as membrane lipids, proteins, and nucleic acids via reactive oxygen species (ROS) . In briel powder . Yang etl powder . The prol powder . Gettingl powder .Pearl powder is a beneficial source of calcium . Xia et 3(PO4)2 or calcium sulphate (CaSO4) which have good biocompatibility but low osteogenic activity [Bone tissue engineering using osteoinductive scaffolds is an emerging approach to bone regeneration. Pearl possesses a unique block-and-mortar hierarchical structure and is potentially a good bone repair material as it has a higher osteogenic activity than HA or nacre . At presactivity . In one activity . High-reactivity ,64. Nacractivity ,65. The activity . Lamgharactivity . Asvanunactivity . These aactivity .Freeze-drying of chitosan-hyaluronic acid (C-HLA) with nano-pearl powder (NPP) produced a scaffold that successfully imitated the composition and structure of bone . The hydZhang et al. fabricated PCL composite scaffolds with various concentrations of pearl powder (30–80 wt.%) with 3D printing . The porDai et al. impregnated natural pearl powder into polylactic acid (PLA) scaffold and observed that HA deposition on PLA/pearl composite increased by up to 15 wt.%, particularly at the beginning of mineralization , suggestIn the work reported by Bai et al., pearl powder was mixed with poly (PHBV) and a composite nanofiber scaffold was prepared from the mixture via electrospinning . Mineral4 composite scaffolds via 3D printing accompanied by a hydration process [4 as a biomaterial [3 which buffers the pH of bone growth sites [4 scaffolds displayed uniform intertwined macropores (~400 mm) with high porosity. The scaffolds also exhibited improved compressive strength (~8 MPa), which is closely comparable to that of natural bone and twice the strength of a plaster-of-Paris, CaSO4 scaffold (∼3 MPa). These pearl/CaSO4 scaffolds possess strong apatite-forming potential, promoted the spread and differentiation of rBMSCs, and enhanced expression of associated osteogenic genes. Notably, the micro-computed tomography and histology of essential rabbit femoral condyle defects implanted with these scaffolds indicated osteogenic potential with fresh bone found after 8 weeks  monofilaments to enhance the biocompatibility of mesh used in pelvic reconstruction . SEM ima process . There amaterial . Pearl pth sites . Pearl/C 8 weeks .The use of resorbable devices for osteosynthesis is an emerging area of focus. Nacre was shown to support bone apposition without triggering an inflammatory response . It was Drug-charged implantable scaffolds can potentially be used for bone cancer chemotherapy, in combination with surgery . ElectroThe use of mother of pearl and pearl powder in traditional Chinese medicine as a cosmetic additive or food ingredient has a long and rich history. In this paper, we have briefly discussed the opportunities, developments and challenges in applications of the use of pearl powder in the treatment of various biomedical issues. Contemporary research of these natural materials has identified their potential biomedical applications with encouraging preliminary findings. Much more study is needed to delineate the possible attributes and mechanism of action of pearl powder for skincare and in medicine. However, a fundamental challenge to this development is that commercial pearl powder is often variable in its consistency and commonly adulterated with low-cost shell powder. Analytical methods are needed to differentiate pure pearl powder from adulterated material. Progress has been made in this respect using microscopic infrared reflectance spectroscopy. Given sufficient spectroscopic data, principal component analysis and partial least squares can provide confidence in distinguishing and categorizing pearl powder samples. This technological progress will be particularly important for the implantation of pearl powder in biomedical applications. As there is no formal attempt has been done to standardise the analytical protocols for the quality assessment of pearl powder among the manufacturers and researchers working on this subject, the meaningful comparison of data becomes extremely difficult. Even most fundamental characteristics of pearl powder can be determined by conventional analytical methods, the correlations between these methods and characteristics of pearl powder are poorly understood. In brief, pearl powder shows promise as an additive for wound healing and tissue engineering to promote cell growth on polymer scaffolds, but reliable identification, quantification and characterization of this complex mixture of components are important to gain a better understanding of its biochemical mechanism of action and expand its potential in biomedical application. Many questions and issues remain open and stimulate future research and innovative solutions. By combining the advantages offered by conventional techniques with the versatility of new processing techniques, new ways for scaling pearl powder production can be envisaged. Nevertheless, a substantial effort is required to eventually translate the exciting properties of pearl powder into a technological and commercial reality.


Combined chemoradiotherapy is the standard of care for locally advanced solid tumours. However, systemic toxicity may limit the delivery of planned chemotherapy. New approaches such as radiation-induced prodrug activation might diminish systemic toxicity, while retaining anticancer benefit. Organic azides have recently been shown to be reduced and activated under hypoxic conditions with clinically relevant doses of radiotherapy, uncaging pazopanib and doxorubicin in preclinical models with similar efficacy as the drug, but lower systemic toxicity. This approach may be relevant to the chemoradiation of glioblastoma and other solid tumours and offers potential for switching on drug delivery from implanted devices. The inclusion of reporters to confirm drug activation, avoidance of off-target effects and synchronisation of irradiation with optimal intratumoral drug concentration will be critical. Further preclinical validation studies of this approach should be encouraged. Concurrent delivery of chemoradiotherapy (CRT) is the standard of care for many locally advanced solid tumours, including head and neck, lung, urological, gastrointestinal, cervix, sarcomas and brain . The rat2 and progressively increased with lower oxygen concentrations  [Under conditions of chronic hypoxia, organic azides can be enzymatically reduced to amines, and this has been exploited to release a fluorophore, for imaging. Compared with normoxic conditions, increased fluorescence was initially observed at 3% O0.1% O2) . The effOverall, the possibility for using an external X-ray beam to uncage functional groups  which are common in existing anticancer agents, in a hypoxic environment, provides a new paradigm of spatial and temporal control over the treatment of solid tumours. Although the reported organic azido compounds were not specifically targeted to tumours, RT offers a significant degree of targeted activation. If prodrug activation can be highly controlled with synchronised localised CRT, tumour cell kill can be maximised while minimising systemic toxicity.The potential for RT activation of metal complexes  , 8 and mAlthough the dominant mechanisms of action for PDT  may depend on individual cancer , PDT-X iThe principal application of the RT-activated organic azide approach is likely to be settings where delivery of optimal dose and duration of conventional CRT is limited by systemic toxicity see Fig. . It is pA limitation of the RT-activated organic azide prodrug approach is the treatment of micrometastatic spread. There might be a role for achieving cytoreduction in the primary tumour, where systemic toxicity prevents further chemotherapy, and the potential for therapeutic gain and reduced radiotoxicity from RT-induced prodrug activation at therapeutic or sub-therapeutic doses. Specific development of prodrugs of potent DNA damage repair (DDR) inhibitors such as platinum compounds, DNA-dependent protein kinase (DNA-PK) and poly-ADP ribose polymerase (PARP) inhibitors, where the current treatment is RT alone (e.g. stereotactic ablative body radiotherapy (SABR) for NSCLC and oligometastases) is likely to be highly beneficial. This might be to either increase tumour control (same RT dose), or to reduce RT dose without compromising tumour control. This may also potentially increase the immunostimulatory effects of RT , since tPrevious attempts to develop orally available prodrugs  has demonstrated the potential for non-linear and highly variable absorption pharmacokinetics , which wThe recently reported doxorubicin azido prodrug demonstrated a dose-dependent response , with 60A typical CRT course might consist of RT of 2 Gy daily (M-F), over a 6-week period, with chemotherapy administered on 3 days during this period. Maximising the benefits of the RT activation approach will have to address the substantial clinical complexity of scheduling prodrug delivery before each RT session with pulses of intravenous chemotherapy. This raises the possibility of either reducing frequency of the RT sessions , or daily RT sessions but with a shorter treatment cycle.The development of RT-activated organic azide prodrugs could be an important addition to the armamentarium of CRT for some solid tumours, irrespective of hypoxic status. Such RT-activated prodrugs might reduce local and systemic toxicity. Inclusion of drug activation reporters, and synchronisation of irradiation with optimal intratumoural drug concentration will be important. We encourage further preclinical validation studies of this approach.


Common iliac artery aneurysm (CIAA) is complicated by ilio-iliac arteriovenous fistulas (IIAVF), which is rare but fatal and require prompt diagnosis and appropriate treatment. As open repair is associated with high morbidity and mortality, endovascular therapy is considered appropriate for treating an IIAVF.A 76-year-old male patient who developed an IIAVF as a complication of ruptured CIAA, requiring immediate surgical repair presented to the hospital with hemodynamic instability.Computed tomography angiography and conventional angiography revealed an IIAVF.Endovascular therapy was selected to reduce the risk of morbidity and mortality. As the angiogram after the first endovascular aneurysm repair with stent-grafting showed contrast medium filling in the aneurysm sac, right common iliac vein, and the inferior vena cava, an aortic extension cuff was inserted into the right common iliac vein to close the orifice on the venous side.The right lower leg edema and discomfort were resolved immediately after the procedure, with the vital signs remaining stable. Computed tomography performed 6 months postoperatively showed patent stent-grafts of the artery and vein, with no evidence of IIAVF and endoleak.IIAVF following CIAA rupture can be repaired successfully by stenting of the common iliac vein with an aortic extension cuff. For successful endovascular repair, the vein side of the fistula tract should be excluded with a stent-graft to block the backflow into the aneurysm sac. Therefore, in such cases, pulmonary embolism may occur because of distal embolization of the thrombus or atheroma in the aneurysm sac. Even anuria or hematuria may occur because of the functional renal damage due to venous congestion. If the condition worsens due to hemodynamic instability or multiple organ failure, emergency surgical intervention is required.The presentation of IIAF may vary depending on its size and location; symptoms including abdominal pain, bruits and thrills, lower leg edema, and unilateral leg ischemia occur because of the changes in the artery-to-vein flow due to venous hypertension. This may ultimately induce a high cardiac output and, subsequently, cause heart failure.Although early diagnosis and appropriate treatment are crucial to attaining good outcomes and preventing exacerbation of the condition, clinical practice guidelines for effective treatment remain unestablished as IIAF is extremely rare with limited relevant published data. Traditionally, surgical intervention has been the primary treatment choice, but less invasive endovascular treatment is performed when anatomically or technically suitable. Herein, we report the case of an IIAVF following CIAA rupture treated successfully by endovascular aneurysm repair (EAVR) using an aortic extension cuff on the CIV.2A 76-year-old male patient presented to the emergency department (ED) with a sudden onset of right lower leg edema and mottled skin color change that started 8 hours prior Fig. . At admiLaboratory workup revealed a hemoglobin level of 11.4 g/dL; creatinine level, 1.40 mL/dL ; and normal electrolytes, liver enzymes, and acid-base balance. Troponin-T was slightly elevated to 0.036 , but no changes were observed on the electrocardiogram. The N-terminal prohormone of brain natriuretic peptide (NT-proBNP) was elevated to 686 pg/mL .Computed tomography (CT) angiography showed right CIAA measuring 54 mm in maximal diameter and an early filling of the contrast medium into the right CIV and inferior vena cava (IVC) in the arterial phase Fig. . The aneUnder general anesthesia, with a percutaneous approach using a suture-mediated closure system  we performed a pre-close technique on both femoral arteries. Embolization of the right internal iliac artery (IIA) was attempted but it failed due to the fast blood inflow from the right CIA to the right CIV and a rigid stenosis at the opening. Hence, EVAR followed by surgical right IIA ligation was decided to be performed if any type II endoleak was identified. The stent-graft deployment was started: an aortic stent-graft system  (25–14 mm × 103 mm) was inserted and a left iliac limb stent-graft (16–24/124 mm) was deployed. Because right IIA embolization was not performed, we deployed 2 iliac limb stent-grafts  extending up to the right external iliac artery.An angiogram obtained after stent graft insertion still showed the contrast medium filling in the aneurysm sac, right CIV, and IVC Fig. A. Thus, The right lower leg edema and discomfort were resolved immediately after the procedure, with the vital signs remaining stable. The patient recovered without complications and was discharged on day 7 of hospitalization. CT performed 6 months postoperatively showed patent stent-grafts of the artery and vein, with no evidence of IIAVF and endoleak Fig. . Current3As an IIAVF occurs rarely after CIAA, selecting its appropriate treatment strategy is difficult, especially since the clinical presentations widely vary and could be life-threatening. Furthermore, the treatment differs depending on anatomy, pathology, and availability of treatment options at each healthcare institution.,9,10 However, the critical aspect of open repair is the bleeding control strategy in the venous orifice. Huang et al introduced the method of controlling bleeding in an aneurysm sac by digital compression or balloon catheter insertion through the fistula tract for inflation and direct closure of the suture. However, failure to achieve proper proximal and distal control after opening the aneurysm sac causes heavy bleeding, which hinders direct suture repair. To control the bleeding on the venous side of the IIAF, extensive dissection up to the iliac vein is necessary to expose the proximal and distal aspects, which may worsen the risk of hemorrhages/hematomas. Moreover, complete hemostasis cannot be achieved through balloon catheter inflation, and calcification of the aneurysm wall hinders suturing. Eventually, conventional open repair increases the morbidity and mortality as the operation time and bleeding risk increases, which prolongs the postoperative recovery period. As reported, aneurysm rupture leads to a high mortality rate of 52%.,3Open repair using a prosthetic graft was regarded as the gold standard.,11,12 This complicated condition is considered difficult to treat by conventional EVAR since it cannot block the blood flow from the vein to the aneurysm sac. The alternative solution to this is by graft stenting in the vein, but this can only be considered in traumatic or iatrogenic injury-related cases.–15 In aneurysm-associated IIAVF cases, backflow occurs from the vein of the IIAVF into the aneurysm sac even after blocking the blood flow with a stent-graft, which may cause sac enlargement or delayed rupture. Thus, theoretically, a type II endoleak occurs and stent-grafts must be placed on both sides around the iliac artery and vein defects.With advances in endovascular techniques, less invasive endovascular repair has been performed when anatomically and technically suitable. They performed emergency EVAR without vein stenting in their patient who developed an IIAVF due to CIAA rupture. The CT from when the patient experienced a rapid exacerbation following their no-stent EVAR showed type IIIa endoleak caused by component separation. In that report, the continuous blood flow into the aneurysm sac through the fistula tract was attributed as the contributor of component separation. Furthermore, the patient experienced complete resolution of symptoms after relining of the arterial site and venous stent-grafting. Thus, theoretically, no hemodynamic changes were expected within the aneurysm sac due to the low pressure on the venous side. Nevertheless, venous stent-grafting should be performed to completely occlude the fistula orifice if possible. It must be noted that cases with long-term follow-up after vein stenting have not been reported.Rishi et al introduced the venous stent-graft placement strategy to prevent sac pressurization in IIAVF caused by CIAA rupture.The diameter and length of the available stent-grafts are factors that need consideration. The recommended diameter of iliac stents is 14 to 16 mm for general cases of stenosis and occluded lesions. However, an AVF distends the diameter of the iliac vein due to high flow; thus, the exact diameter of a suitable vein stent cannot be easily identified. While preoperative CT is useful as a reference, measuring the iliac vein diameter in the intraoperative angiogram may help. Because the vein distends further after resuscitation in cases of intraoperative hemodynamic instability, oversizing should be considered in such cases. They reported that the venous site was excluded, but the stent-graft was occluded due to the pressure from the iliac aneurysm. Therefore, they recommended the use of short stent-grafts with radial force on the vein side. We used an aortic extension cuff  that covered a short range of the CIV and was suitable for the patient's venous diameter. This was possible because the AVF site had been accurately identified on CT and angiography. Thereafter, we could fit the CIV length accurately and oversized appropriately, resulting in an adequate radial force, which was vital for maintaining the patency of vein stenting. Thus, surgeons should use various types of stent grafts considering the CIV diameter or range of the AVF site.Accordingly, iliac extension limbs or aortic extension cuffs, or thoracic stent-grafts can be used, after selecting the diameter or length of the available stent-graft, especially in emergencies. Sueyoshi et al reported a case involving a long stent limb placement (16–20/124 mm) into the venous side because the exact AVF site could not be located during EVAR.One factor that reduces the flow into an aneurysm sac is occluding the venous blood flow and backflow from the internal iliac artery. Usually, IIA embolization is first attempted, but once an IIAVF is formed, the large flow volume from the iliac artery to the iliac vein causes contrast filling of these vessels on the angiogram, which hinders the identification of the IIA orifice. This occurs more frequently in cases involving IIA stenosis. Thus, when the patient is hemodynamically unstable, a stent-graft should be implanted from the aorta to the external iliac artery at the AVF site with venous stenting to occlude the fistulous channel followed by surgical IIA ligation if needed.4CIAA complicated by IIAVF can be successfully treated after judging the anatomical and pathological condition of the patient, as well as the available infrastructure at healthcare institutions. While there are many challenges in the treatment of such IIAVF cases because of the varied clinical presentations, endovascular treatment can be appropriately planned since it is less invasive and effective. Successful endovascular treatment of IIAVF following CIAA rupture is dependent on controlling the bleeding on the venous side and, if anatomically suitable, excluding the vein side using an aortic extension cuff is one viable treatment option.www.editage.co.kr) for English language editing.I would like to thank Editage (Data curation: Young Sun Yoo.Investigation: Young Sun Yoo.Methodology: Young Sun Yoo.Visualization: Young Sun Yoo.Writing – original draft: Young Sun Yoo.Writing – review & editing: Young Sun Yoo.


The diagnosis of Bezold’s abscess can be challenging especially when craniofacial malformations imply facial and cervical morphological asymmetries. In addition, craniofacial malformations might predispose to the occurrence and atypical diffusion pathways of suppurative processes originating from abnormally developed temporal bone structures.A 30-year-old female presented with a left laterocervical swelling, worsening over time. The female was affected by Goldenhar syndrome. CT and MRI were performed.CT revealed a dysmorphic os tympanicum and a deep cervical abscess in continuity with its cavity. Drainage of the cervical abscess was performed but a subsequent brain MRI detected a large cholesteatoma that was removed with left lateral petrosectomy.Radiology has a crucial role in the diagnosis and planning of the treatment of Bezold’s abscesses, particularly in syndromic patients. MRI, in this case, helped in diagnosing the presence of the cholesteatoma and consequently appropriately approach the surgical removal. The resulting phenotype is considerably heterogeneous: common features are auricular abnormalities  and hemifacial microsomia.6 The most severe cases also present with eye or spine involvement .7 The spectrum of abnormalities might encompass the central nervous system, cranial nerves and ipsilateral inner ear, internal carotid artery and salivary glands.8–10 Because of external ear malformation, these patients might be more vulnerable to regional infections, especially when a congenital cholesteatoma is present.11 In the most severe forms of facial asymmetry,6 local signs of inflammation, tympanic membrane bulging or hyperemia and otorrhea might not be evaluable or partly hindered by the altered anatomy of the temporal bone. Consequently, infectious processes might be easily overlooked, and severe complications might arise.11Oculo-auriculo-vertebral spectrum 12 due to erosion of the medial mastoid tip at the level of the digastric groove, determining the diffusion of the suppurative process into the posterior cervical space.13 First described in 1881 by the German otologist Friedrich Bezold, this complication has become less common most probably because of early management of mastoiditis with antibiotics as well as with modern imaging tools.14 Acute otitis media, cholesteatoma or necrotizing external otitis are the most common causes of mastoiditis associated with Bezold’s abscess.14,15 Typical clinical presentation is a floating laterocervical swelling frequently but not constantly accompanied by pain, fever, hearing loss, otorrhea and retroauricular swelling.14 Radiology has a crucial role in identifying the suppurative process, in delimiting its extension in the cervical spaces and in recognizing possible features, such as a coexistent cholesteatoma, mastoid bone erosions or peculiar anatomical characteristics that condition the subsequent surgical management.Bezold’s abscess is a well-defined through extreme rare complication of mastoiditisHerein, we report on a patient affected by Goldenhar syndrome who developed Bezold’s abscess as a complication of an os tympanicum cholesteatoma.A 30-year-old female was admitted because of left laterocervical swelling, worsening over time despite protracted large spectrum antibiotic therapy. The female was affected by Goldenhar syndrome, with left craniofacial microsomia, aural atresia, microphthalmia and multiple cervical vertebral anomalies . Her medAt admission, head and neck CT  showed s−2) tissue inside the os tympanicum with milder peripheral contrast enhancement compared to the residual abscess’s walls  cholesteatoma. They present a partially subverted anatomy, and their facial asymmetry might interfere with the identification of the laterocervical swelling leading to a delay in the diagnosis.6 As the association of cholesteatoma and mastoiditis might become an impending condition, in craniofacial malformation patients early imaging with bone CT and diffusion-weighted MRI sequences are pivotal for a prompt, tailored and adequate treatment.These observations strengthen the diagnostic role of MRI, both in characterizing cervical soft tissue involvement and in recognizing the typical DWI hyperintensity of the cholesteatomatous tissue. A proper MR and CT imaging protocol appears to be pivotal, especially in patients with craniofacial malformations, where cholesteatomas are more common and often implicated in suppurative processes.The presence of craniofacial malformations might impair the early detection of a neck abscessA Bezold’s abscess may arise from the os tympanicumAn underlying cholesteatoma might favour Bezold’s abscess formation.For the best surgical management, the diagnostic work-up should include both bone CT and diffusion-weighted MRI.


We present the case of a 75-year-old female with acute embolic cerebral infarction caused by a fail-implanted venous port catheter system in the left subclavian artery.A 75-year-old woman presented to our emergency room after acute onset of a right-sided hemiparesis and dysarthria. Within 2 days after admission, she developed a left-sided hemiparesis, ataxia with concordant gait disturbance and incoordination of the left upper limb. DWI-MRI showed acute multiple infarcts in both cerebral and cerebellar hemispheres. Laboratory examination, 24-h Holter electrocardiography and transthoracic echocardiography provided no pathological findings. Further examination revealed an arterially fail-implanted port catheter, placed in the left subclavian artery with its tip overlying the ascending aorta, as the source of cerebral embolism.This is the first case report of thromboembolic, cerebral infarction due to a misplaced venous port catheter in the subclavian artery, emphasizing the imperative need for a thorough diagnostic workup, when embolism is suspected but cannot be proven at first glance. Central venous port systems are widely used in modern medicine. The usual complications comprise of local or systemic catheter-related infections, local thromboses with swelling of extremities and malfunction due to mechanical abnormalities. Careful insertion and standardized procedures to ensure correct placement of the port catheter system are vital to avoid periprocedural complications.We present the first case of a woman with cerebral infarction caused by a failed implanted venous port catheter system residing in the subclavian artery.A female in her 70s was admitted to our hospital with the suspected diagnosis of acute ischemic stroke after acute onset of a right-sided hemiparesis. Amongst others, the medical history revealed a short bowel syndrome after ascendojejunostomy with the necessity of diet therapy and parenteral nutritional supplementation via venous port catheter. The percutaneous implantation of the venous port catheter in the left subclavian vein was performed in a different hospital a week before hospital admission. Over recent years, several ports had already been removed due to catheter-associated complications like lumen occlusion and port-related infections. The port currently inserted had not been used for parenteral nutrition previously.The patient underwent a standard acute stroke diagnostic workup consisting of non-contrast cranial computed tomography (CT), a 24-h Holter electrocardiography without indications for atrial fibrillation and transthoracic echocardiography revealing normal heart structures. The laboratory results from the patients blood were all within normal paramters, especially the coagulation test . Extracranial and intracranial duplex sonography indicated marginal atherosclerosis without hemodynamically effective stenoses.Secondary prophylaxis with antiplatelet (aspirin) and statin medication was initiated. Low-dose enoxaparin was also prescribed to minimise the risk of venous thrombosis. The patient benefited considerably from regularly performed physical therapy, and arm function improved during the first 48 h. Two days after admission, the patient showed an acute onset weakness of the left upper and lower limb, including mild cerebellar signs on the left side.Brain magnetic resonance imaging (MRI) 2 days post stroke showed multiple small ischemic lesions bilaterally in the cerebellum as well as in subcortical and cortical areas of the cerebral hemispheres, affecting the supplying territory of arteries in the anterior and posterior circulation on both sides Fig. . With reBefore injecting a small amount of saline solution into the port, the patency of the system was verified by cautious aspiration. As the blood returning to the syringe appeared moving pulsatile, an arterially misplaced port catheter was suspected.The patient was transferred immediately to a vascular surgery department. Conventional angiography showed an arterially placed catheter Fig.  which waThe patient’s neurologic status improved missing clinically overt signs of recurrent stroke, and she was able to be transferred to a rehabilitation center after implantation of a new central venous access port device. The patient recovered completely without any neurologic deficit 3 weeks after admission to our hospital.Central venous ports are considered a routine device for long-term administration of chemotherapeutic agents, transfusion blood sampling, repetitive antibiotics and intravenous hydration, or parenteral nutrition, mostly to cancer patients or—as in our case report—chronically ill patients , 2.The use of a central venous port was initially described by Niederhuber et al., who reported the first implantation in 1982 via the subclavian vein .A reliable implantation technique and regular maintenance of the venous port systems is indispensable in order to avoid catheter and port-related complications, such as catheter obstruction, venous thrombosis, infection and mechanical complications. In the literature referring to larger patient cohorts, the indicated complication rate ranges from 10.4 to 15.1% , 7.A broad variety of methods exist to implant a port catheter: Surgeons cut down or percutaneously puncture the forearm and upper-arm peripheral veins, the subclavian, or jugular vein, whereas radiologists perform minimally invasive venography, ultrasound, or fluoroscopy guidance of the subclavian or jugular vein , 8.A careful insertion of the catheter system and skilled management should decrease the incidence of complications. The early diagnosis of arterial misplaced catheter can be obtained by the color of blood, the pressure and pulsatile backflow observed through the catheter, blood gas analysis and the postinsertion chest x-ray , 4, 8–10Clinical findings and laboratory samples help to identify infectious complications, including local and catheter-related blood stream infections. Venous thrombotic complications usually manifest as arm or facial swelling, cyanosis, venous distension, and superficial venous collateralization. Mechanical complications include catheter malposition, occlusion or fragmentation, port damage and fibrin sheath formation. These complications lead to system malfunction, usually discovered earlier and, in consequence, system explantation and reimplantation may occasionally be required. Further complications, such as infusion pump dysfunction or human error, can also occur and are difficult to distinguish from mechanical complications , 9–11.Such cases will have a rarity value. In current literature, no case has been described wherein an arterially displaced port catheter was left in situ a period of time, causing cerebrovascular events such as embolic cerebral infarction. Cerebrovascular symptoms caused by infusion of parenteral medication and nutrition via venous catheter were described only in a few cases .Neurologic symptoms presented in temporal association with the insertion of a newly placed venous central port catheter might help to ensure the correct diagnosis before irreversible damage occurs. Intermittent embolization of small clots formed on the tip of the catheter following shear-induced platelet aggregation might be the possible pathophysiologic mechanism responsible for focal neurologic symptoms in the presented case. Nowadays, the arterially placed catheter would have probably been detected earlier, as CT-angiography is now routinely used in stroke patients, according to national stroke guidelines. This is due to the fact that CT-angiography helps to identify patients with large-vessel occlusion that might benefit from thrombectomy.This case report highlights the symptoms of embolic cerebral infarction related to an arterially misplaced port catheter and emphasizes the imperative need for a thorough diagnostic workup when embolism is suspected but cannot be proven by standard diagnostic stroke workup.Ultrasound guidance to localize the anatomic conditions of preferred veinPost insertion chest x-ray with critical evaluationBlood gas analysis and testing the catheter with about 100 ml of crystalloid solution running via gravity through newly placed catheterReevaluation of catheter conditions in case of neurologic symptoms occurred with initiation of newly inserted catheter and after initiation of infusion.To avoid catastrophic complications of misplaced venous port symptoms, the following steps should be taken:


Blood-brain barrier (BBB) disruption following ischemic stroke (IS) contributes to hemorrhagic transformation, brain edema, increased neural dysfunction, secondary injury, and mortality. Brain endothelial cells form a para and transcellular barrier to most blood-borne solutes via tight junctions (TJs) and rare transcytotic vesicles. The prevailing view attributes the destruction of TJs to the resulting BBB damage following IS. Recent studies define a stepwise impairment of the transcellular barrier followed by the paracellular barrier which accounts for the BBB leakage in IS. The increased endothelial transcytosis that has been proven to be caveolae-mediated, precedes and is independent of TJs disintegration. Thus, our understanding of post stroke BBB deficits needs to be revised. These recent findings could provide a conceptual basis for the development of alternative treatment strategies. Presently, our concept of how BBB endothelial transcytosis develops is incomplete, and treatment options remain limited. This review summarizes the cellular structure and biological classification of endothelial transcytosis at the BBB and reviews related molecular mechanisms. Meanwhile, relevant transcytosis-targeted therapeutic strategies for IS and research entry points are prospected. Stroke is one of the leading causes of death and disability worldwide, and ischemic stroke accounts for approximately 87% of cases . BBB disWhile the existence of a unique barrier between the central nervous system (CNS) and peripheral circulation was hypothesized by Paul Ehrlich and his student Edwin Goldmann over a century ago , the preob/ob mice paired with t-MCAO as a model of enhanced BBB breakdown and reported microvasculature structural changes of the BBB via two and three-dimensional EM [ob/ob and ob/− control mice showed increased endothelial vesicles at 4 h and 24 h after ischemia, with no significant change in TJ structure or expression of related proteins in isolated microvessels visualized by EM [ob/− controls, ob/ob mice had increased lesion volumes and BBB permeability to IgG and HRP. Correspondingly, ob/ob mice displayed increased counts of striatal endothelial vesicles at 4 h, localizing at the most severe region of BBB disruption [Employing two-photon microscopy to visualize dynamic TJ changes, Knowland et al. observed fluorescent tracer leakage across the BBB in transgenic mice with fluorescently labeled TJs following induction of the transient middle cerebral artery occlusion (t-MCAO) stroke model. It was noted that BBB leakage began as early as 6 h after ischemia but TJs only displayed profound structural defects after 48 h. Conversely, the number of endothelial caveolae and rate of transcytosis increased beginning at 6 h after ischemia, indicating stepwise impairment of transcellular, followed by the paracellular, barrier mechanisms which contribute to BBB deficits in stroke  Figure . Moreoveional EM . Vesselsed by EM . Comparesruption . In ligh+/+Mfsd2a and Mfsd2a−/− mutant mice, with either increased or decreased transcytosis, mirrored the aforementioned relationship between BRB integrity and transcytosis levels; elevated transcytosis resulted in delayed BRB sealing and mice with suppressed transcytotic activity displayed earlier BRB sealing [Endothelial transcytosis in the CNS is practically negligible under basal conditions, but a prominent feature following acute neurologic injury. Specifically, the rapid increase of this process in IS brings forth the exact role and contribution of endothelial transcytosis in the formation of a sound functional barrier. Delineating this fundamental question in normal development and physiology provides fundamental understanding and key mechanisms dysregulated in stroke. To this end, Chenghua Gu’s team employed a zebrafish model to investigate the involvement of endothelial transcytosis in a vertebrate BBB development. In leveraging this powerful optically transparent model organism with live EM, Gu et al. visualized in real-time the dynamic formation of the zebrafish BBB . Early d sealing . TherefoRecognizing the normal interplay between transcytosis and barrier formation/integrity alludes to the pathological ramifications when either these conjoined processes are disturbed following IS; the increase in CNS endothelial transcytosis following stroke, and their earlier increase and independence of TJ disruption in the acute phase of IS. Preclinical interventional studies further show that suppression of the BBB endothelial transcytosis in the acute phase of stroke preserves TJ expression, which conserves BBB integrity and brain homeostasis . These iThe remainder of this review briefly summarizes the cellular composition, biological classification, and molecular regulatory mechanisms which underlie BBB endothelial transcytosis. Meanwhile, relevant transcytosis-targeted therapeutic strategies for IS and research entry points are prospected.The “sandwich” structure of the BBB determines that its constituent components, the ECs, pericytes, and astrocytes, are structurally adjacent and functionally coordinated to control the influx and efflux of substances into the CNS. Pericytes envelop ECs and are embedded within the basement membrane to facilitate extensive and reciprocal intrasignaling. Several influential studies have shown that the precise ratio of pericytes to ECs is critical to BBB integrity; pericytogenesis ,17,18,19Transcytosis is the vesicular trafficking of molecules between the luminal and abluminal cell membranes. Macromolecules are first endocytosed or internalized by vesicles on one side of the cell, trafficked in vesicles, and then exocytosed or released on the other side of the cell. It remains poorly understood what type of vesicles and different transcytosis mechanisms are involved. How vesicle formation and transcytosis are maintained at low levels in brain ECs is also unknown. The current consensus postulates that transcytosis in CNS endothelial cells can be divided into two categories: receptor-mediated transcytosis (RMT), in which ligand-receptor binding mediates endocytosis such as transferrin and insulin ,22, and The two known major endocytic pathways at the BBB are clathrin-mediated and caveolae-mediated . ClathriCaveolin and cavin play a key role in the formation of caveolae. Cavin is an adaptive protein that forms oligomers crucial for membrane bending, and caveolin is an integral membrane protein that binds to cytosolic cavin to form small vesicles ,30. CaveCav-1−/− mice compared with age-matched wild-type (WT) controls [−/−Cav-1 mice [Increasingly, studies associate Cav-1 with the permeabilized BBB following various pathological conditions, including IS. It has been found that Cav-1 expression increases in the rat cortical at 12 h after hypothermic brain injury, preceding the decreased expression of occludin and claudin-5 at 48 h . Moreovecontrols . Knowlancontrols . Moreove−/− mice .Cav1−/− mice compared to WT controls [Cav1−/− mice also displayed enhanced hemispheric swelling, lesion volume, and worsened neurological outcomes compared to WT controls. These worsened outcomes coincided with reduced neovascularization and modified astrogliosis, without the formation of proper glial scarring around the lesion at 3 days post injury [Cav1−/− mice. Gu et al. recently showed that, unlike other vascular segments of ECs in the CNS, arteriolar ECs have abundant caveolae and actively relay signals from the CNS to smooth muscle cells through a caveolae-dependent pathway to realize vasodilation, which is independent of the endothelial NO synthase (eNOS)-mediated NO pathway [These findings endorse the notion that upregulation of Cav-1 is functionally related to an increase in BBB permeability via increased endothelial transcytosis. Given the important role of caveolae and Cav-1 in early BBB breakdown following IS, they are attractive targets of modulation to attenuate BBB dysfunction, and thus theoretically provide the downstream benefits of arresting edema formation and infiltration of peripheral immune cells, crucial elements for improving stroke outcomes. Still, it is worth mentioning that studies have also reported that Cav-1 deficiency can aggravate the injury of IS. Work by Knowland et al. supports the divergent detrimental effects of deficiency, showing increased lesion volumes in controls . Hirt ancontrols ,38. Furtt injury , decreas pathway . In pati pathway .The seemingly contradictory results reviewed from these studies indicate the complex pathways which participate in transcytosis, further complicated by disease states. The divergent effects of Cav-1 in stroke point to its pleiotropic capacities, including cell growth, differentiation, cholesterol trafficking, and cellular senescence . The useSignificant differences differentiate barrier characteristics and vesicle numbers between central and peripheral ECs; whether BBB ECs express a specific set of genes that govern the formation and maintenance is a major outstanding question. Again, innovative work from the Gu team illuminated answers to this question. Gu et al. identified over 200 highly expressed differential genes in cortical ECs that are barely expressed in lung ECs . Among tIncreased BBB permeability is correlated to hemorrhagic transformation, which represents the main limitation of rt-PA thrombolytic therapy in IS. Presently, tPA remains the only approved therapeutic treatment available for IS patients; however, strict inclusion criteria and a limited therapeutic time window limit its ubiquitous usage for patients. Creative strategies to overcome these realities would substantially alter the treatment approach for IS and benefit many patients yearly. Suzuki et al. showed that the transient increase of BBB permeability is related to the endothelial transcytosis in the ischemic area, which is partially regulated by vascular endothelial growth factor (VEGF) ,47. They®  targeted endothelial transcytosis by significantly reducing the number of cortical endothelial caveolae and expression of Cav-1. Targeting this mechanism rescued BBB-integrity after cerebral ischemia in rats [d-glucoside can significantly inhibit the decrease of Cav-1 protein in hypoxic and glucose-deprived cerebral microvascular ECs [Various intervention strategies derived from natural medicines or traditional therapies have demonstrated targeted effects on CNS endothelial transcytosis in IS. Ping et al. identified increased numbers of cortical endothelial caveolae at 3 h and 6 d after t-MCAO, accompanied by a corresponding increase in Cav-1 expression. Treatment with compound Cerebralcare Granule in rats . Alterna in rats . Using t in rats . Zou et  in rats . In vitrular ECs . ResultsIncreasing studies have shown that caveolae-mediated transcytosis increases as an immediate response in the acute stage of IS. This response may be a precursor of irreversible nerve injury, while some scholars have innovatively proposed that it may also serve as a new and promising opportunity to deliver therapeutic drugs into the brain ,56. NanoFocused ultrasound (FUS) is a new therapeutic method that can lead to a transient, targeted and reversible opening of the BBB. Deng et al. determined that FUS treatment in combination with microbubble cavitation can enhance BBB permeability through caveolae-mediated transcytosis by upregulating the Cav-1 expression and number. This Cav-1-mediated transcytosis may cooperate with other transport pathways to induce the opening of the BBB . Pandit In all, research on caveolae-mediated BBB endothelial transcytosis in acute IS is on the ascendance. The increase of transcytosis is considered a precursor to the permeabilized BBB in the acute phase of IS but may also represent a new opportunity to deliver therapeutics to the injured brain. The physiological and pathological significance, related regulatory mechanisms, specific targets, and targeted CNS drug delivery all remain to be defined, while clinical translation of this exciting process depends on further preclinical and clinical research. It is already known that caveolae-mediated BBB endothelial transcytosis is a transient and reversible process that does not destroy the essential structure of the BBB. This process is accompanied by the occurrence and change of transcytosis at the BBB endothelium such as the shift with aging from efficient ligand-specific receptor-mediated transcytosis to inefficient nonspecific caveolae transcytosis during blood-brain transport . Recent


COVID-19 and Fontan physiology have each been associated with an elevated risk of venous thromboembolism (VTE), however little is known about the risks and potential consequences of having both.A 51 year old male with tricuspid atresia status post Fontan and extracardiac Glenn shunt, atrial flutter, and sinus sick syndrome presented with phlegmasia cerulea dolens (PCD) of the left lower extremity in spite of supratherapeutic INR in the context of symptomatic COVID-10 pneumonia. He was treated with single session, catheter directed mechanical thrombectomy that was well-tolerated.This report of acute PCD despite therapeutic anticoagulation with a Vitamin K antagonist, managed with emergent mechanical thrombectomy, calls to attention the importance of altered flow dynamics in COVID positive patients with Fontan circulation that may compound these independent risk factors for developing deep venous thrombosis with the potential for even higher morbidity. COVID-associated coagulopathy has recently been described  and COVIA 51-year-old male with tricuspid atresia, status post atriopulmonary Fontan at 6 years of age with subsequent urgent surgical conversion to an extracardiac Fontan Fig. , the patUpon transfer, his INR was down-trending, but remained supratherapeutic at 6.6, and computed tomography venogram (CTV) demonstrated bilateral deep venous thrombosis from the iliac to calf veins, excluding the inferior vena cava (IVC). There was no evidence of pulmonary embolus. Physical exam was notable left lower leg findings pitting edema, purple discoloration Figs. , 4, decrWhile the differential diagnosis of limb ischemia in the setting of COVID-19 infection includes arterial thrombosis, the demonstration of venous outflow obstruction Fig. , violaceFollowing multidisciplinary evaluation, the decision was made undergo an emergent transvenous thrombectomy to salvage the left leg. Given the inflammatory nature of the clot and development of PCD in spite of supratherapeutic INR, mechanical thrombectomy was felt to be the most appropriate course of action  as opposBilateral popliteal vein access was obtained for catheter directed mechanical thrombectomy using the ClotTriever® system . Popliteal and Fontan pressures were 19 and 17 mmHg, respectively; pulmonary angiography did not demonstrate any emboli. Five thrombectomy passes of the left lower extremity from the popliteal vein to the IVC and four passes of the right leg successfully restored flow from the bilateral popliteal veins to the IVC Fig.  and yielHe experienced epistaxis while on heparin, that was treated with nasal packing. There was evidence of post-pneumonia fibrosis  (Fig. 2CPatients with a Fontan circulation are at higher risk for thrombus formation . The sysPCD requires aggressive therapy for limb salvage , but resPresently, the most effective anticoagulation strategy for patients with COVID-19 related thrombosis is unclear. Our patient developed no further thrombosis while on heparin, though notably this change was made after the COVID pneumonia was already improving.Patients with comorbidities that place them in higher-risk categories for various disease states, such as Fontan circulation, are potentially at higher risk of complications from COVID-19. PCD is a rare but devastating complication, and must be recognized early and treated aggressively; mechanical thrombectomy may be indicated to expedite restoration of venous outflow, particularly when anticoagulation may be contraindicated. Pulmonary fibrosis secondary to COVID-19 may lead to increased pulmonary vascular resistance, which may further complicate the long-term course of Fontan patients in particular. Despite the presence of acute lung injury and extensive deep venous thromboses, our patient did not require mechanical ventilation or develop decompensated heart failure, reflecting a reassuring degree of resilience of this patient’s Fontan circulation in the face of COVID-19 infection.


Imatinib, a selective inhibitor of the breakpoint cluster region (BCR)-ABL kinase, is the poster child for targeted cancer therapeutics. However, its efficacy is limited by resistance mutations. Using a quantitative bioluminescence resonance energy transfer assay in living cells, we identified ABL kinase mutations that could cause imatinib resistance by altering drug residence time. Protein phosphorylation, catalyzed by protein kinases, is a critical signaling process essential to life. Dysregulation of this post-translational modification drives the pathogenesis of many human diseases including autoimmune, cardiovascular, nervous system disorders, and cancer. It was originally thought that targeting members of the kinome with adenosine triphosphate (ATP)-competitive small molecules was infeasible due to high sequence conservation of the ATP-binding site. The discovery of imatinib, an inhibitor of the breakpoint cluster region (BCR)-ABL oncoprotein, upended this myth. BCR-ABL, a fusion between the breakpoint cluster region and ABL kinase, is the major driver of leukemogenesis in chronic myelogenous leukemia (CML). Imatinib reduced the mortality rate of CML by 80% within a decade of its approval. Consequently, imatinib became the poster child for specific kinase inhibition and spawned an entire field of kinase-targeted drug discovery. Roughly a third of modern drug discovery efforts are focused on small-molecule modulators of kinases.2 Imatinib achieves its high specificity in part by binding preferentially to an inactive conformation of ABL kinase, whereas the more promiscuous inhibitor dasatinib binds to the active conformation. Furthermore, conformational dynamics affect both the drug-binding process3 and dissociation process,4 thereby providing a vulnerability for therapeutic intervention and small-molecule design.Active kinases adopt strikingly similar poses when catalyzing phosphotransfer with their substrates. However, kinases are not just static phosphorylation machines; they are dynamic proteins that interconvert between a large ensemble of inactive conformations and a more conserved active conformation.Kd, the standard measure of a lead compound’s efficacy, into the rate constants for drug binding to the target (kon) and dissociation from the target (koff) may be more relevant for non-equilibrium in vivo systems. Residence time, the reciprocal of the drug dissociation rate constant, describes the lifetime of the drug-target complex and has been shown to be a superior predictor of in vivo potency in several systems.5 Drug dissociation rates have been of particular interest to kinase inhibitor development since the discovery of BIRB796, a reversible p38α mitogen-activated protein kinase inhibitor with an unusually long estimated residence time of >1800 hours.6 The analysis of 270 kinase inhibitors against 40 kinases revealed an increasing number of slow-dissociating compounds when transitioning from early/preclinical compounds to late stage and FDA-approved compounds.7 Kinetic selectivity, where the drug-target complex has a longer half-life than off-target-drug complexes, is an emergent strategy to achieve specific kinase inhibition. For example, we modified a series of aminopyrimidines to create inhibitors with much longer residence times for focal adhesion kinase over the closely related proline-rich tyrosine kinase 2.8Drug action in the body is tied to dynamic processes such as absorption, distribution, metabolism, and excretion (ADME). Therefore, dissection of the equilibrium binding constant This led us to ponder: if chemical modifications to inhibitors can improve their efficacy by prolonging residence time, could the reverse be true for mutations in kinases that reduce inhibitor efficacy by reducing residence time?9 we used NanoBRET,10 a bioluminescence resonance energy-transfer (BRET) technique that quantitatively assesses target engagement in live cells, to test the affinity and dissociation kinetics of imatinib and dasatinib against a library of 94 ABL kinase domain mutations. These mutations were observed in CML cases with resistance to imatinib therapy. Surprisingly, we found that two-thirds of mutations significantly reduce drug affinity while one-third of mutations retain similar or tighter affinity to wild-type (WT). This posed the question: how could 31 mutations that have similar binding affinity to imatinib cause imatinib resistance? We found mutations at three sites, N368S, V299L, and G251E with significantly faster imatinib dissociation rates. These sites are frequently altered in other kinases and cancer types: there are nine mutations across eight kinases reported for N368, three mutations across three kinases reported for V299, and 13 mutations across eight kinases reported for G251. We further validated the observed effect of the N368S mutation in vitro and proposed a mechanism of its increased dissociation rate using molecular dynamics simulations.In our recent study, Lyczek et al.,In contrast to thermodynamic mutations that decrease the imatinib binding affinity, kinetic mutants increase the drug off-rate and on-rate while maintaining a similar affinity to WT . While tThis study represents the first comprehensive comparison of the effect of disease-relevant mutations on drug affinity and binding durability in live cells. Mutations at these sites are present ubiquitously throughout the kinome, which suggests that kinetic resistance to structurally selective kinase inhibitors may be a widespread mechanism. We envision that similar investigations of mutations in other drug-target pairs will uncover a broader role for binding kinetics in drug resistance and impact clinical standards of care.


Applications of small-angle X-ray scattering (SAXS) in structural biology are reviewed. A brief introduction of the SAXS basics is followed by the presentation of the structural features of biological macromolecules in solution that can be assessed by SAXS. The approaches are considered allowing one to obtain low resolution three-dimensional (3D) structural models and to describe assembly states and conformations. Metrics and descriptors required for the assessment of model quality are presented and recent biological applications of SAXS are shown.  •Small-angle X-ray scattering (SAXS) is a powerful technique in structural biology.•SAXS allows to study biological macromolecules in close to native solutions.•Low-resolution models of proteins, nucleic acids and complexes can be obtained.•Atomic models can be integrated using hybrid analysis approach.•SAXS is applicable to partially or completely disordered macromolecular states. SAXS is sensitive to both ordered and not-ordered features in the sample and it does not require crystallization, fixation, or vitrification procedures. In a SAXS experiment A, a collh)−1 see ); a concvs.r exceeds the maximum intramolecular distance  ele shape . Machinele shape .Fig. 2A.The SAXS profile of a macromolecule can be described by a finite set of values spaced at m SHANUM ) excludem SHANUM D.A useful presentation of SAXS data is the Kratky plot of  version    can be eMap test  allows fab initio reconstructions, and comparison of the results of repeated reconstructions is often useful. Metrics such as the normalized spatial discrepancy (NSD) are applied to the set of solutions, yielding a stability measure (average NSD significantly exceeding unity indicates ambiguous solutions). For dummy-atom modelling, inspection of the total spread region (TSR) and of the most populated volume (MPV) reveal the regions of increased variability and also features common to the different models , such as SREFLEX  Fig. 4BBFig. 4B.Intrinsically disordered proteins, or those containing significant unfolded fragments, are best modelled considering co-existing ensembles of conformers. The first approach developed to this end, the ensemble optimization method (EOM) , selectsXS (EROS ), which Mycobacterium tuberculosis (ab initio modelling well reproduces the overall toroidal shape of the complex and the SAXS data can also be neatly fitted by the scattering computed from the crystal structure (Saccharomyces cerevisiae (Examples of recent applications of biological SAXS utilizing various analysis approaches are given in rculosis A, the abtructure . Fig. 5Brevisiae . Here, trevisiae  is preserevisiae . The strrevisiae .Fig. 5A.Summarizing, SAXS is a versatile tool in structural biology, offering broad possibilities for sample characterization in solution at the nm scale. Thanks to straightforward integration with other techniques, SAXS plays an increasing role in the validation and study of conformations and assemblies of biological macromolecules in solution.Stefano Da Vela: Writing - original draft, Visualization. Dmitri I. Svergun: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Complication rates associated with intercalary allograft reconstruction may be reduced by maximizing tenants of allograft reconstruction. Intercalary allograft reconstruction using a hemispherical reaming technique for graft-host interface may increase surface contact areas, provide intimate contact between surfaces, and equally distribute pressure subsequently decreasing risk of nonunion. The purpose of these case reports was to present short-term results for limb salvage using this novel technique for two young, active duty military members who returned to full-impact activity. Allograft reconstruction has the advantage of maintaining bone stock in younger patients and may avoid the late complication of aseptic hardware loosening seen in prosthetic reconstructions. However, lack of incorporation of the intercalary bone graft can present several unique problems. Nearly half of patients experience a delayed union, defined as no radiographic union by 1 year after surgery, and graft fracture has been reported to occur in up to 30% of patients.7 When compared with transverse osteotomies, the sigmoid and step-cut provide 74% and 44% greater contact surface area, respectively.5 The concave-convex reamed interface provides more than doubled the surface contact area, when compared with a transverse cut.4 Concave-convex osteotomy was also more effective in comparison with transverse osteotomies in evenly distributing pressure across the graft-host interface, which may prevent stress concentration and subsequent fracture.5 Clinical results in the literature are limited to one technique article reporting the successful implementation of the reaming technique in two allograft patients.8Several techniques have been developed to promote union including transverse cut, step-cut, sigmoid cut, and concave-convex reaming; however, there is a paucity of literature comparing union rates and time to union of concave-convex reaming versus former methods.We hypothesized that concave-convex reamed intercalary allografts provide a biomechanical advantage over traditional osteotomies and may provide a durable enough construct to return to full-impact activity. We present the clinical results of concave-convex intercalary allograft reconstruction in two active duty military patients, with return to full duty, after diaphyseal tibia resection for primary bone sarcoma with intermediate-term results.A 27-year-old active-duty male infantry soldier presented with right leg pain after a physical fitness test and a presumed diagnosis of a tibial stress injury. Initial radiographs demonstrated a lucent lesion of the mid-diaphyseal tibia with biopsy and staging consistent with an isolated high-grade osteosarcoma of the tibia Figure . He then6 the allograft was first cut transversely with 1 cm of additional length at both the proximal and distal ends to allow reaming. Complementary hemispherical reaming of the allograft tissue was done using a reverse reamer from the Equinoxe shoulder resurfacing system (Exactech); reamer sizes are available in 3 mm increments from 38 to 53 mm. Normal saline irrigation was used for lubrication and to aid in temperature reduction during reaming. Allograft fit was grossly assessed frequently in vivo. The graft was then secured with hybrid locking-compression plating.The tumor was resected using standard transverse cuts based on preoperative measurements, and reconstruction was undertaken after intraoperative pathology confirmed disease-free margins. The proximal and distal aspects of the host tibia were then shaped with 53 mm and 44 mm acetabular reamers, respectively. The fresh-frozen, size-matched allograft  was prepared on the back table and held with bone reduction forceps during preparation. Similar to the technique described by Wilkes et al,9 with several changes. The patient remained non–weight bearing for 6 weeks postoperatively followed by an additional 6 weeks of toe-touch weight bearing. After 12 weeks, the patient was advanced to 50% weight bearing and began therapy with the use of a weight-limiting, low-impact treadmill (AlterG). Patients were progressed to full weight bearing once either the patient was pain free, and radiographs demonstrated osseous bridging or 16 weeks had elapsed since surgery. Full-impact activity was not permitted until there was osseous bridging at both the graft interfaces.Compression of the plate and a graduated progression of weight bearing was believed to allow for a stress healing response without overloading the initial avascular, distal ends of the allograft while allowing for soft-tissue healing. Subsequently, our postoperative protocol required monthly radiographs and limited weight bearing until union as described by Ortiz-Cruz et alOsseous bridging was noted at the proximal tibial metaphyseal interface 12 weeks postoperatively and the distal diaphyseal interface 28 weeks postoperatively. A computed tomography (CT)-guided biopsy of the distal host bone tibia was obtained 32 weeks postoperatively to evaluate for a possible recurrence noted on radiographs. The histology demonstrated benign, fibrous tissue. The CT scan allowed for additional characterization of the host allograft interface confirming healing of the proximal junction with near circumferential union of the distal junction except for the anteromedial 20%.The patient was advanced to full weight bearing at 16 weeks and was running with the assistance of an Intrepid Dynamic Exoskeletal Orthosis (US Army) at 36 weeks postoperatively. At the last follow-up (33 months), the patient had returned to full active duty including marching with a full combat-load (70 pounds) without pain or evidence of construct failure 11-13 resulting in amputation rates of up to 20%15 which we would likely encounter more frequently with a larger patient cohort and was not related to our surgical technique.We observed decreased time to union at the meta-diaphyseal versus diaphyseal-diaphyseal host-allograft interfaces as expected by previous studies8 when compared with amputation.16 However, in a subset of young patients, amputation may be a more attractive option to allow full weight bearing and impact activities. Attempts at activity after allograft reconstruction should only be permitted after a thorough risk-benefit discussion. It should be noted that our patients would not have been able to continue in their current career with either amputation or activity restriction necessitating a trial of full activity including running, jumping, and carrying loads.Functional outcomes in patients after diaphyseal tibial resections are promising after intercalary allograft reconstructionLimitations in this study include patient volume, lack of long-term follow-up, or a comparison group. In addition, patient selection may have had a notable impact on their outcome because these both were physically fit, highly functional individuals with access to state-of-the art rehabilitation. The contribution from the surgical procedure is hard to quantify because our time to union was not unexpectedly different from historical controls and we had no comparison group; however, at the final follow-up, we demonstrated that two adult patients with intercalary tibial allografts were able to maintain a previously unreported level of function with this surgical technique. Further research is required before widespread adoption of this technique and would include larger prospective, comparative studies including multiple conventional graft preparation techniques. Reconstruction options must continue to address this gap in the treatment armamentarium to provide patients with a durable yet functional limb that supports the aspirations of the young and active but will endure into old age.To the best of the author's knowledge, this is the first to report of adult sarcoma patients returning to running and impact activity after intercalary allograft reconstruction. The contribution of concave-convex reamed junctions to these patients' outcomes cannot be determined from this limited case series, but the durability demonstrated in these two constructions should encourage future prospective, controlled studies to determine the ideal construct for highly active patients undergoing bone sarcoma resection.