Composites are macroscopic combinations of chemically dissimilar materials preferred for new high-tech applications where mechanical performance is an area of interest. Mechanical apprehensions chiefly include tensile, creep, and fatigue loadings; each loading comprises different modes. Fatigue is cyclic loading correlated with stress amplitude and the number of cycles while defining the performance of a material. Composite materials are subject to various modes of fatigue loading during service life. Such loadings cause micro invisible to severe visible damage affecting the material’s performance. Mode I fatigue crack propagates via opening lamina governing a visible tear. Recently, there has been an increasing concern about finding new ways to reduce delamination failure, a life-reducing aspect of composites. This review focuses on mode I fatigue behaviours of various preforms and factors determining failures considering different reinforcements with respect to fibres and matrix failures. Numerical modelling methods for life prediction of composites while subjected to fatigue loading are reviewed. Testing techniques used to verify the fatigue performance of composite under mode I load are also given. Approaches for composites’ life enhancement against mode I fatigue loading have also been summarized, which could aid in developing a well-rounded understanding of mode I fatigue behaviours of composites and thus help engineers to design composites with higher interlaminar strength. Fibre reinforced composites are now used for emerging structural application areas such as aerospace, automotive, wind turbine, defense area, marines, civil engineering and leisure equipment as replacements for conventional metallic materials ,2,3,4,5.Compared to other damage types, delamination growth is a prevalent life-reducing failure mode for composite structures causing the separation of adjacent plies ,4,7,8. TA damage tolerance design approach assumes that defect already exists in the structure and permits valuation of the time to failure. Interlaminar fatigue characterization of composites is essential to providing valuable input for damage-tolerant design philosophy. It is employed in the testing for the cyclic performance of materials since three dimensional (3D)-woven composites are used in lightweight structural components, beams, structural joints, and wind turbine blades. Apart from this, one of the most critical applications of 3D composites is in the Boeing Dreamliner airliner (in the inlet fan blades of the GenX gas turbine engines). In all these applications, composites are exposed to cyclic or variable loading conditions, so knowledge about fatigue response, including delamination resistance to the initiation and growth of cracks, is typically important.a = crack length (delamination length);N = number of cycles;da/dN = fatigue crack growth rate;A, m =material constants for curve-fitted power law.Delamination propagates under the three pure modes: mode I (opening mode), mode II (sliding mode) and mode III (tearing mode) or as a combination of these . FatigueAccording to the Paris law given in Equation (1), the cyclic crack evolution rate depends only on the stress intensity factor range , Compression–Compression (C-C), Tension–Compression (T-C), bending, thermal and mixed mode loadings ,22. VariIt is assumed that crack starting and crack proceeding are the two stages that occur during fatigue. Extreme crack starting points are obtained first in cyclic loading, and a large part of the fatigue life is crack proceeding at high fatigue stress generally known as low cycle fatigue (LCF). In high cycle fatigue (HCF), the fatigue crack must be initiated after a significant number of total fatigue cycles. In metal, fatigue initiation is obtained at the surface where defects or stress concentration are most likely to be found. In composites, fibre/matrix heterogeneity of matrix gaps can produce the defect point and thus it is not unusual to find the subsurface fatigue origins.Fibre fracture, matrix fracture and fibre–matrix delamination are the three dominant fatigue failure modes observed in unidirectional single layer composite. The fibre strength has a considerable effect on fibre fracture. The high stiffness of the fibres can delay the matrix fracture because a lesser amount of deformation passes to the matrix. Delamination occurs when the fibre–matrix interface is simulated by weak fibre/matrix bond strength. In brief, various damage mechanisms interact, hence it is quite challenging to analyse the fatigue failure in composites. This is true for unidirectional as well as multidirectional textile composites.Considering these challenges, in this paper we have provided a review of the investigations that have been made on mode I fatigue loading of fibre reinforced composite. Sufficient information on fatigue behaviour and failure modes of various preform structures is given, which could help to adopt a defect tolerant approach both in design stage and in residual life estimation. Several parameters that affect the performance of composites under mode I fatigue loading such as temperatures, load rate and effect of fibre bridging are described briefly. Methods to improve the fatigue performance of composites are also summarized.2. Y. Nakai and C. Hiwa [Most of the FRP composites are made from laminates. Among various composite structures, composite laminates’ fracture is the most common.  C. Hiwa  examined C. Hiwa .Kenane, M. et al. ,37, condVauthier et al.  studied J. Hoffmann and G. Scharr analysed the fatigue resistance of composites having two different geometric shape pins (rectangular and circular shape). According to the Paris curves, the rectangular z-pins were more efficient in growing delamination resistance of quasi-isotropic composites unlike the circular z-pins. In unidirectional composites, when the cross section of the pins changed, it slightly affected the mode 1 fatigue resistance of the z-pins composite.Multidirectional laminated composites are those in which the layers are arranged at different angles to produce multidirectional stiffness properties for broad area uses such as quasi-isotropic laminates as shown in These microcracks combine and propagate in out-of-plane direction until they reach a favourable interlaminar interface. The development of composite structure requires extensive knowledge of the movement process, particularly similarities and differences between fatigue and quasi-static delamination movement.Peng et al.  studied sistance .T Chocron et al. studied the fracture and fatigue delamination growth in multidirectional laminated composites under mode I fatigue loading. A Paris-type relation was obtained by fatigue tests of DCB specimens, which describes the delamination growth rate where a is the delamination length and ere used . Banks-Sere used  studied et layup . Delaminet layup .Gong et al.  studied Due to the greater mechanical properties, 3D woven composites have gained a lot of attention in many application areas such as aerospace, civil engineering, marine, transportation and automotive industries ,46,47,482) [The minimum value of cyclic stress intensity factor (00 J/m2) . This re00 J/m2)  used metLadani et al.  developeFibre failure and matrix failure are the two major damage processes often found in composite during cyclic loading ,61,62. TFailure of fibres in composite static or fatigue failure can be categorised as tensile and compression fibre failure . Fibre pFailure in the matrix during fatigue loading can be divided into two failure modes, the first one is matrix failure in a ply known as inter-fibre fracture and the second one is matrix failure in between piles known as delamination. There has remained always a great interest in developing the better fibre–matrix interfacial properties . Inter-fA microcrack begins to develop when the applied load is strong enough to induce fibre failure . These mFibre failure, matrix failure, delamination and fibre–matrix separation are among the primary failure modes of laminates. The failure condition of the polymer matrix composites could be brittle  or ductiIn case of fibre reinforced polymeric composites, fibre bridging performs a vital role in fatigue delamination resistance. After a certain growth of delamination length, the crack development decreases significantly due to crack shielding effect of bridging fibres . The briA single Paris resistance curve has been used to evaluate the fatigue crack expansion, which is found to be ineffective in case of bridging fibres, as the bridging fibres can vastly increase delamination resistance . A theorHere, Yao et al.  also anaTo describe the impact of significant fibre bridging on fatigue delamination of multidirectional laminates, both the Paris relation and the modified Paris relation (with a modern similar variable) has been used. Bridging fibre seems to be typically an artefact of unidirectional laminated composites’ testing. It happens in laboratory DCB tests, but hardly detected and reported for delamination in actual working conditions. It makes sense that to design a structure for functioning life, the required cyclic delamination data should be conservative in that it eliminates fibre bridging . The autCoronado et al.  investigA. Sjogren and L.E. Asp  examinedAndrouin et al.  investigAndersons et al.  introducYao et al.  analysedArgüelles et al.  studied Apart from experimental work, there are some numerical studies on Mode I fatigue delamination of composite. Fracture models are mandatory to forecast fatigue life and create appropriate scrutiny intervals to found and fix the delamination before it becomes life-threatening or exceeds the residual strength of the component . VariousTo analyse the crack propagation process and to extract the strain energy release rate at crack tip, a technique known as “Virtual Crack Closure” (VCCT) technique has been used. The method is based on the supposition that the energy be found  as(3)ΔEThe energy release rate can be obtained asTo apply VCC technique, the expected crack growth path must be predicted. This method is commonly used in 2D finite element models with one or two crack fronts. The use of extended finite element model (XFEM) permits simulating intralaminar and interlaminar crack development. Contrary to the VCCT method, XFEM does not need a pre-defined crack path. This technique is effective in simulations where crack growth is not already known . EnricheThe cohesive zone model (CZM) approach is another widely used method in modelling crack propagation in interface. CZM has the capability to model the delamination onset and propagation without defining the initial defect, unlike the fracture mechanics method ,55,83. IAfter reaching the maximum interfacial stress, the damage occurs. In fatigue loading conditions, there are two possible approaches of modelling interface element damage accumulation . The firi. A similarity was found between the CZM and experimental results by Moura [In cycle-by-cycle loading/unloading fatigue damage formulation, tracking the accumulation of permanent damage and simulation of progressive damage in the softening region of the CZM is achieved byby Moura . When coFor CZM defined with loading envelope strategy, the softening law was regarded as a function of number of cycles. For each model time-step, the fatigue damage parameter is updated using. Harper  establisF. Teimouri  extendedSkvortsov used shell elements instead of 2D elements or solid elements to model fatigue delamination propagation in composite laminates. Loading envelope strategy combined with algorithm of fatigue damage is defined in a CZM model. Delamination length and fatigue response of composite under fatigue loading were simulated. The proposed approach was found efficient and sufficiently accurate. The fatigue life of the examined specimen was accurately predicted. The delamination length dramatically increased after 60,000 cycles. The fatigue and quasi-static response of composite were obtained with proposed model.The numerical simulation method has been increasingly accepted as a valuable tool for predicting the damage initiation and propagation. It has been an essential technique in fatigue life prediction, failure mechanisms investigation and fatigue strength optimization of composite.There are many testing techniques (destructive and non-destructive) and specimen configurations to determine the mode I fatigue delamination of laminated composites such as the double cantilever beam test (DCB) ,42,83,92DCB configuration as shown in The DIC is one of the most widely used techniques to determine the mode I fatigue delamination in composites. Zhu et al.  used theMaillet et al. studied the impact of frequency on cyclic mode I fatigue delamination growth and compared it with standard tests accomplished at 10 Hz frequency. The vibration resonance machine has been used to measure the mode I fatigue delamination rate of composite . A dynamOn the other hand, Brunner et al.  found thAnother method known as interlayer of interleaf is the use of toughened resin system to improve the interlaminar fatigue of carbon fibre reinforced composites. This system involves adding thermoplastic or thermoset particles to the resin system ,30. InteStevanovic et al.  used intHowever, particle interleaved composites have some drawbacks such as decrease in in-plan stiffness and strength (15–20%)and increase in laminate thickness (~20%) and potential decrease of Tg . ElectroStructural properties such as high modulus, high strength and good creep resistance of fibre reinforced polymeric composites can be enhanced by infusing nanoparticles. Graphene, carbon nanofibres (CNFs), spherical nanoparticles, carbon nanotubes (CNTs)are all outstanding candidates for reinforcement which are dispersed in matrices in smaller amount and present improvements in properties of polymeric materials .The trend of cellulose nanofibres (CeNFs) has much increased due to their higher mechanical properties, excellent sustainability, production, and demand for the growth of composites ,100,101.Ladani et al.  found thRomhany et al.  investigMany studies ,109,110 Z-pinning or Z-binder yarns are well-known approaches for improving the fatigue delamination resistance of fibre reinforced composites . Z-direcJ. Hoffmann and G. Scharr  analysedSimilarly, z-pins were used to analyse the mode I fatigue resistance of carbon–epoxy laminated composite by incorporating them in through-thickness direction . Zhang ePingkarawat and A.P. Mouritz  analysedDenis et al.  reportedAnother study  reportedZ-pins made of low fatigue resistant material such as copper collapse quicker under mode I fatigue loading than z-pins comprised of high fatigue resistant materials such as carbon fibre, and consequently this reduces their capability to enhance the cyclic delamination properties. In a similar study, Abbasi et al.  quantifiTherefore, we can summarize that mode I fatigue properties of z-pinned reinforced composites can be tailored by the right choice of z-pins content, size (length and diameter) and material. The polymeric composite interfaces can be changed with thermoplastic layers or PA66 fibres to enhance their crack growth resistance and fatigue life. For example, CFRP interface was modified by addition ofPA66 nanofibres of approximately 520 nm diameter, which decreased the crack propagation rate up to 30 times . The craExceptional properties with low weight make composite materials ideal for the era. However, several factors influence composite materials’ service life, among which fatigue is a prominent phenomenon. Composite material parameters are viable in governing mode I fatigue. Textile preform architectural variations govern mode I fatigue behavioural differences among composite materials. Multidirectional laminated composites perform better in mode I fatigue than unidirectional reinforced composites. Z-yarns in through-thickness direction significantly enhance mode I fatigue behaviour of 3D woven reinforced composites. Though 3D reinforced composites have excellent mode I fatigue performance, very limited studies reported their fatigue behaviour under mode I cycling loading.Both compression and tensile failure modes are involved in mode I fatigue loading. Reinforcement fibre failure occurs due to imbalanced arrangements and debonding in compression and tensile modes, respectively. In contrast, matrix fails due to inter-fibre fractures initiated by interlaminar stresses. Although environmental factors such as temperature and aging have a pronounced effect on fatigue life of composites, nevertheless very limited research is found on the effect of environmental factors on mode I fatigue. A small number of studies on mode I fatigue reports the effect of temperature, i.e., temperature is influential in defining fatigue life. The effect of aging on mode I fatigue delamination resistance is not currently captured. Numerical studies also present models for predicting the mode I fatigue life of composite materials.Testing techniques, including cantilever beam tests, digital image correlations, X-ray tomography, and acoustic emissions detections are used to predetermine fatigue behaviours of composite materials’ service life. In a nutshell, tremendous work has been done in the literature regarding mode I fatigue behaviours of composite materials, opening several new ways to explore further. Different techniques employed for mode I fatigue life enhancement are fruitful, including resin toughening, z-pins insertion and nano modification of composites using carbon nanotubes, cellulose nanofibres, etc. However, there are discrepancies on the effect by volume or weight content of these nanomaterials and their working mechanisms that need further attention of researchers.


Superior mesenteric arteriovenous fistula is a rare vascular anomaly often presenting with sequelae of portal hypertension, heart failure, or mesenteric ischemia. This report describes a patient with a previously unidentified superior mesenteric arteriovenous fistula who presented with variceal bleeding, thought to be the leading cause of mortality associated with this condition. Although this patient was initially referred for a transjugular intrahepatic portosystemic shunt procedure, following a thorough review of her clinical history and imaging, she instead underwent embolization of the arteriovenous fistula likely responsible for her symptoms.A 75-year-old woman with a past surgical history of extensive small bowel resection presented with active variceal bleeding requiring transfusions. She was referred to vascular and interventional radiology for transjugular intrahepatic portosystemic shunt procedure; however, her clinical presentation was inconsistent with cirrhosis. This prompted a further review of her imaging, which identified a superior mesenteric arteriovenous fistula as the probable etiology of her varices. This fistula was subsequently embolized with a vascular plug and follow-up upper endoscopy at 1-month demonstrated complete resolution of her varices.This report highlights a potential etiology of variceal bleeding in the acutely ill patient. Through a thorough consultation, the patient described here was able to avoid a procedure with the potential to cause catastrophic consequences, and instead receive the appropriate treatment for an uncommon condition.Level 4, Case Report. The primary etiologies of superior mesenteric arteriovenous fistulas (SMAVF) are traumatic, iatrogenic, and congenital . She had also developed ascites within the preceding four years, presumed to be secondary to parenteral nutrition-associated liver disease.The patient was resuscitated with seven units of packed red blood cells and four units of fresh frozen plasma. Upper endoscopy visualized grade 3 esophageal and grade 2 gastric varices. Balloon tamponade was performed with a Linton tube and the patient was transferred to a tertiary care hospital on octreotide and a proton pump inhibitor. VIR was consulted to evaluate the patient for a TIPS procedure; however, several findings were inconsistent with a working diagnosis of portal hypertension due to cirrhosis.The patient did not have typical cirrhotic risk factors  and fibrosis was not seen on a liver biopsy 4 years prior, so long-term use of TPN was the primary factor that would predispose her to cirrhosis. These considerations, along with a lack of cirrhotic features on an abdominal computed tomography angiography (CTA) scan, indicated that cirrhotic liver disease was unlikely. Closer inspection of the abdominal CTA scan revealed abnormal hyperenhancement and dilation of both the superior mesenteric vein (SMV) and portal vein in the arterial phase. Further review confirmed the presence of a SMAVF distal to the intestinal branches of the superior mesenteric artery (SMA) Fig. .Fig. 1CWe proceeded with embolization of the SMAVF to reduce portal pressure. A 6-F 25 cm sheath was placed in the right common femoral artery. A 5-F Cobra 1 catheter  and 2.4-F Progreat microcatheter  were used to cannulate the SMV across the fistula from the SMA . At her 2, 3, 12, and 16-month postprocedural follow-up visits, the patient’s ascites had resolved (weaned off diuretics) and she denied any recurrence of gastrointestinal bleeding. Four-phase liver protocol computed tomography scan at 16-months post-procedurally demonstrated no further evidence of a portosystemic shunt, a patent portal venous system, resolution of ascites, and no recurrence of esophageal or gastric varices. This patient will continue to be followed in the VIR clinic indefinitely for surveillance of the SMAVF and liver disease management.The management of SMAVF is either surgical or endovascular. The surgical option provides definitive fistula closure, but is also associated with higher perioperative risk (Zhao et al., If the patient had received a TIPS procedure as requested on the initial consult, she would not only be at significant risk for heart failure, but also recurrent variceal bleeding (An et al., In summary, the present case describes a successful SMAVF closure via vascular plug embolization, in a patient presenting with gastrointestinal hemorrhage initially referred to VIR for a TIPS procedure.


How stromal cells control tumor progression is an active area of investigation. Cancer associated fibroblasts (CAFs) have been suggested to both limit, or promote cancer dissemination in various contexts, but a systemic understanding of stromal influence in determining cancer metastasis is lacking. This study takes a closer look at the diversity of fibroblasts that emerge in different metastatic stages of PDAC , a cancer type with high stromal infiltration. Specifically, we found that the CAFs respond to the growing (but yet not metastasized) tumor by transition to a myofibroblast like stage, potentially resisting cancer invasion. As the cancer disseminates, these fibroblasts are nearly entirely replaced by highly diverse set of subpopulations with distinct functions. We have previously advanced a framework, termed Evolved Levels of Invasability (ELI), explaining the vast differences in cancer metastasis and placental invasion among mammals. We found that PDAC’s metastatic transition and stromal trespass is accompanied by the downregulation of pro-resistive and the upregulation of pro-invasability ELI signatures in CAF subpopulations. Cancer-associated fibroblasts (CAFs) are now appreciated as key regulators of cancer metastasis, particularly in cancers with high stromal content, e.g., pancreatic ductal cell carcinoma (PDAC). However, it is not yet well understood if fibroblasts are always primed to be cooperative in PDAC transition to metastasis, if they undergo transformation which ensures their cooperativity, and if such transformations are cancer-driven or intrinsic to fibroblasts. We performed a fibroblast-centric analysis of PDAC cancer, as it transitioned from the primary site to trespass stromal compartment reaching the lymph node using published single-cell RNA sequencing data by Peng et al. We have characterized the change in fibroblast response to cancer from a normal wound healing response in the initial stages to the emergence of subclasses with myofibroblast and inflammatory fibroblasts such as signatures. We have previously posited “Evolved Levels of Invasibility (ELI)”, a framework describing the evolution of stromal invasability as a selected phenotype, which explains the large and correlated reduction in stromal invasion by placental trophoblasts and cancer cells in certain mammals. Within PDAC samples, we found large changes in fibroblast subclasses at succeeding stages of PDAC progression, with the emergence of specific subclasses when cancer trespasses stroma to metastasize to proximal lymph nodes (stage IIA to IIB). Surprisingly, we found that the initial metastatic transition is accompanied by downregulation of ELI-predicted pro-resistive genes, and the emergence of a subclass of fibroblasts with ELI-predicted increased invasibility. Interestingly, this trend was also observed in stellate cells. Using a larger cohort of bulk RNAseq data from The Cancer Genome Atlas for PDAC cancers, we confirmed that genes describing this emergent fibroblast subclass are also correlated with lymph node metastasis of cancer cells. Experimental testing of selected genes characterizing pro-resistive and pro-invasive fibroblast clusters confirmed their contribution in regulating stromal invasability as a phenotype. Our data confirm that the complexity of stromal response to cancer is really a function of stage-wise emergence of distinct fibroblast clusters, characterized by distinct gene sets which confer initially a predominantly pro-resistive and then a pro-invasive property to the stroma. Stromal response therefore transitions from being tumor-limiting to a pro-metastatic state, facilitating stromal trespass and the onset of metastasis. The understanding of cancer metastasis as a cell autonomous disease of tumor cells has changed in the last decade to being a collective outcome of cancer cells and the acellular and cellular constituents of the tumor microenvironment . AlthougWe have previously advanced an evolutionary framework, termed the Evolved Levels of Invasibility (ELI), explaining the large differences in the rate of cancer metastasis across mammals, which also correlates with the degree of their placental invasion into the endometrial stroma during pregnancy . ELI posCell-type-specific data within cancers are now beginning to be collected, both at the transcriptomic level by single-cell RNA sequencing (scRNAseq) and single-cell proteomics ,12,13. Hdn genes, which are highly expressed in epitheliochorial species , similar to our previous findings from bulk data in melanoma [up genes, expressed in hemochorial species  [We identified remarkable changes in the fibroblast signaling state during early cancer dissemination, marked by an initial fibroblastic activation responding to the growing primary lesion, followed by a sudden increase in fibroblast heterogeneity as cancer disseminates to neighboring lymph nodes. Recently, using transcriptomic data from endometrial fibroblasts of many mammalian species, we and our collaborators have identified genes and transcription factors that have correlatively changed according to their levels of invasibility . Crucialmelanoma . Furtherrodents) . We confThe multi-tumor single-cell RNAseq data in pancreatic adenocarcinoma were obtained from Peng et al. . We used Seurat  for all cell clustering and differential expression analysis between cell clusters. Tumor and normal samples were co-clustered together, using the method of Stuart et al. . For cluReceptor–ligand partners were obtained from Ramilowski et al. . The strp value < 0.05, and abs(log2FoldChange) > 0.5 for further processing. Upstream regulators and transcription factors were obtained using IPA using the above thresholds for DE genes. All GO and KEGG pathway enrichments were calculated using ClusterProfiler 4.0 [dn and ELIup subnetworks were computed by including protein interactions from Reactome [up or ELIdn gene set was compared with all the gene pairs for all genes outside the ELIup/dn set using a t test. Short Time-series Expression Mining  was used to find statistically significant patterns of gene expression changes across stages [The top genes aligning to endometrial invasability were obtained using the linear regression in Suhail et al. ,20. Geneiler 4.0 . ELIdn aReactome . Gene–ges stages . Optimal cutting temperature (OCT) compound-embedded human pancreatic ductal adenocarcinoma samples were obtained from UConn Health Pathology Biorepository. For representative PDAC samples at pre-metastatic stage IIA, and at the onset of lymph metastasis characterized by stage IIB, we obtained a T2N0Mn/a sample and a T3N1Mn/a sample, respectively, isolated from patients after Whipple resection. The N0 sample was obtained from a de-identified 59-year-old African American, with a tumor size of 3.2 × 3.0 × 2.8 cm with G1 histological grade. From 9 examined lymph nodes, none were found to be positive, suggesting a non-metastatic stage. N1 samples were obtained from a de-identified 67-year-old Caucasian male. From the 13 nodes examined, 5 were involved with metastatic carcinoma. OCT-embedded samples were cryosectioned on glass slides and fixed using 4% paraformaldehyde in PBS solution for 20 min. After washing with PBS twice, samples were permeabilized using 0.2% Triton X-100 for 20 min and washed again with PBS. Thereafter, sections were blocked using blocking buffer (10% goat serum in PBS and 0.1% Triton X-100) for 4 h and incubated with primary antibody in blocking buffer overnight at 4 °C. Unbound primary antibody was washed with PBS 4 times, samples were reblocked using blocking buffer for 1 h, and incubated with Alexa-fluor 488 conjugated secondary antibody in blocking buffer for 1 h. After incubation, samples were washed with PBS 4 times, and were mounted on glass coverslips with antifade solution and imaged. Imaging was performed on Zeiss Observer Z1 microscope with Colibri.2 LED-based fluorescence and Apotome.2 sectional illumination using a 20× objective. Primary antibodies used were: anti-phospho-β-catenin (Ser675) D2F1  and anti-HIF-1a . ANSIA platform was fabricated using methods previously described . BJ and δArea by the length of the initial cancer–stroma interface.Invading cancer cells were imaged using live epifluorescence microscopy every 1 h for 24 h. Invasion fronts were tracked using Region of Interest (ROI) panel in the Fiji software. Underlying anisotropic nanotextured resulted in highly directional invasive fronts, both accelerating the phenomenon, as well reducing dimensionality of the data. ROIs were determined by manual tracing of boundaries, and the normalized extent of invasion determined by dividing total ROIs were converted to a one-dimensional profile of the invasive front, which was smoothed by moving the average over 20 pixels. Smoothed profile was used to calculate the mean signal to either side (40 pixel each) of every point, and a peak was determined when the smoothed profile was larger than either side averages. Consecutive peaks emerged and were determined at the midpoint of the interval. N), and distant metastasis (M) [n = 11 patients) were homogenous and largely constituting a single cluster (cluster 2) , identified with the stages of the cancer progression . We idenasis (M) . t-sne auster 2) B, non-meuster 2) C. As theuster 2) D. Remarkuster 2) E. Transiuster 2) D,E. The uster 2) F. An iniuster 2) G. Because fibroblast subclasses in pancreatic ductal cell adenocarcinoma changed dramatically with the emergence, and then the transition of cancer towards metastasis, we explored the gene expression data further within these clusters. Gene ontology (GO) analysis showed that clusters present in pre-metastatic stages had a higher presence of genes belonging to extracellular matrix (ECM) generation, cell–matrix interaction, as well as hypoxia H. ClusteTo identify the transcription factors which might explain the large differences in gene expression of fibroblast clusters, we calculated the upstream regulators using Ingenuity Pathway Analysis A. SelectIn addition, fibroblasts in cluster 0 show activation of serum response factor (SRF), as well as myocardin-related transcription factor A (MRTFA), both key players in regulating cellular contractility. MRTF-A is well known to control the myofibroblast differentiation of stromal cells , and it Overall, a fibroblast-centric analysis of PDAC progression showed large changes in stromal reaction to cancer as it progresses towards metastasis. Cancer-associated fibroblasts were very different from fibroblasts from a normal pancreas, the latter largely encoding genes associated with physiological functions in the pancreas, indicating either a dramatic shift in the fibroblast state, or more likely a different origin of CAFs. As the tumor grew, the heterogeneity of the fibroblasts actually decreased dramatically (from IB to IIA), and this shift was associated with the emergence of a homogenous class of fibroblasts marked by matrix remodeling phenotype. It would appear that the growth of the tumor resulted in the activation of fibroblasts, resulting in activation of wound healing response, activating canonical pathways associated with matrix production and remodeling. Surprisingly, these fibroblasts were nearly completely replaced by a highly heterogeneous population as the cancer cells trespassed the stroma to reach proximal lymph nodes (Stage IIB). Matrix remodeling phenotype was replaced by either overactivated fibroblasts  or those inhibiting matrix remodeling (cluster 4), vasoregulation (cluster 1), and antioxidant activity (cluster 6). Having identified that PDAC cancers, which are remarkable for their high stromal content and heterogeneity, show large changes in fibroblast heterogeneity in succeeding stages of early metastasis, we directly analyzed the gene expression changes in fibroblasts in each succeeding stage. Gene ontology (GO) and KEGG signaling pathway enrichment analysis of fibroblasts showed the activation of many ontologies associated with matricellular protein assembly . Gene exThe most dramatic effect in fibroblast heterogeneity was observed in the transition of PDAC cancers from IIA to IIB, which is characterized by the first detected stromal trespassing of cancer cells to proximal lymph nodes . GO enriAs the cancer becomes more metastatic spreading to a larger number of lymph nodes (stage III), we found further upregulation of gene sets associated with the secretory cellular machinery , indicatWe identified the genes encoding ligands expressed in fibroblast cells at every stage of cancer, matched to their cognate receptors in other cell types present at the same stage. In normal pancreatic tissue, most interactions by fibroblasts with other cell types indicated their supporting stromal role . These idn genes encode gene products which increase stromal resistance to invasion, while ELIup genes encode gene products which increase stromal invasibility, within the context of the evolved changes in stromal invasibility across mammals  they offer . We have mammals A. Transc mammals B. Using itecture C,D. We fnetworks C,D, suppdn genes in samples from patients transitioning from non-metastatic to early lymph node metastatic cancers  database . We spec cancers E. Most g cancers , includi cancers E. These  cancers . Other ainvasion , but itsdn and potentially pro-invasive ELIup genes show any significant pattern in stage-wise stromal expression. We conducted a Short Time-series Expression Mining (STEM) analysis [dn genes which are more highly expressed in cow and horse stroma, we found a significant pattern of genes which continued to increase expression till stage IIA, and then decreased expression as the cancer trespassed stromal compartment   was strongly correlated with immunosuppressive signature in pancreatic cancers [up signature  I. As the vs. IIB J. Althou cancers   with significantly high enrichment of ELIup genes, suggesting that cluster 4 subtype of fibroblasts may potentially be pro-invasive  emergence of a subclass of fibroblasts with pro-invasable ELIup signature, both data being confirmed in the larger patient cohort data from TCGA.We therefore finally tested if this change in fibroblast diversity across cancer stages showed the enrichment of pro-invasive fibroblasts. As noted earlier, the transition from stage I to stage IIA is characterized by a homogenization of fibroblasts, mostly grouped in cluster 5 in our initial t-sne analysis. As cancer breaches its boundary to trespass through stromal compartment to the lymph nodes, many other clusters emerge , and to a lesser extent cluster 6 and 7 K. We askinvasive L. Intereic state . Finallyic state M. These Stage-wise analysis of heterogeneous fibroblast clusters in PDAC cancer revealed a dynamic stromal environment that changed from resistive to pro-invasive as the tumor becomes metastatic. Based on our theory of Evolved Levels of Invasability, as well as combined gene set enrichment analysis on larger PDAC patient data from TCGA, there was a strong indication that as a tumor becomes larger, fibroblasts are activated with a strong resistive signature, while as it disseminates into proximal lymph node, the stromal trespassing is associated with a marked disappearance of resistive fibroblasts (cluster 5) and emergence of a distinct category of pro-invasive fibroblast subtype (cluster 4). We therefore sought to experimentally test if genes characterizing these clusters indeed confer the said phenotype to fibroblasts in general. We used ANSIA, a method we have previously described, abbreviated for Accelerated Nanopatterned Stromal Invasion Assay, which allows the observation of cancer invasion dynamics into the stromal compartment in an accelerated time frame, owing to the orthogonal arrangement of collagen fibrils mimicking nanotextured ridges underneath the monolayers. Fibroblasts can be genetically perturbed while keeping the fluorescently labeled invasive cancer cells as the constant to specifically test the role of stromal genes in regulating stromal invasability (or resistance) A. We tesTo experimentally confirm the causality of genes characterizing cluster 5 and cluster 4, predicted to be pro-resistive and pro-invasable by our ELI framework, their emergence correlating with the onset of early metastasis, we tested a candidate gene from the key genes characterizing either cluster . We chosIt is now well appreciated that although tumorigenesis may be largely an autonomous function of cancer cells, its progression to a metastatic disease depends on factors beyond the biology of cancer cells themselves, involving many other stromal components ,79. AmonAlthough the changes in fibroblast activation as cancer progresses have been detailed, there is still a lack of coherent understanding of how fibroblasts may orchestrate or assist in the transition of cancer from a non-malignant to a malignant phenotype. Do they actively pull and disseminate cancer cells from the boundary of their primary location, or do they merely allow cancer cells to pass through the stromal compartment by increasing their invasibility? What mechanism induces fibroblasts to change these phenotypes, since these are clearly not inherent properties of normal fibroblasts? Additionally, are all changes in the fibroblasts also cancer induced, wherein cancer is still the active player, recruiting fibroblasts to its own assistance? These are crucial questions, because even if we understand the mechanisms which render CAFs to be assistive in metastasis, the mechanism is still positioned from the perspective of the presumed active player, the cancer cell. What selective advantage does a fibroblast have in allowing cancers to invade into the stromal compartment? If cancer metastasis is a pathological incorporation of other physiological mechanisms through which fibroblasts assist in cancer invasion, could we pose our question from that physiological context? dn genes which are higher in cows, and potentially impart stromal resistance to its fibroblasts, are reduced in human melanoma. These resistive genes, when lost, correlate with increased cancer metastasis and reduced survival.The evolutionary framework, Evolved Levels of Invasibility (ELI), based on a remarkable and large change in placental invasion across mammals identifies the evolved changes in stromal resistance (or invasibility) as a phenotype . This frHere, we have attempted to explore the changes in the fibroblast landscape as cancer progresses from an initial tumor mass through the metastatic cascade. We chose pancreatic cancer as an appropriate model owing to its particularly high stromal content, as well as its lethality. Overall, we found the classical fibroblast response to cancer , with nudn genes (which are expressed higher in bovine and equine stroma) were downregulated in fibroblasts as PDAC cancer transitioned from a pre-metastatic to an early metastatic stage. Notably, this trend was also observed for stellate cells, which are key effectors of tissue fibrosis in the pancreas, although the downregulation of ELIdn genes was observed later (in stage III) in stellate cells. Furthermore, in a larger cohort of PDAC patient data from TCGA, we found that ELIdn genes were reduced in patient samples with early lymph metastasis vs. those with no lymph metastasis, a finding similar to melanoma [dn genes is accompanied by the onset of metastasis. Other analyses further confirmed these patterns. In a somewhat contrasting light, we also found that most top ELIup genes were upregulated with the onset of tumor itself, indicating that the stromal preparation for invasibility is laid earlier than when metastatic events occur, while the resistive defenses are reduced as the cancer cells trespasses the stromal compartment to reach proximal lymph nodes. Finally, an interesting finding with a significant implication for potential therapeutics targeted at stroma is the observation that a particular fibroblast subclass  is both enriched for ELIup genes, and in patients as cancer metastasizes to a lymph. This subclass is characterized by Wnt activation, cyclic AMP driven transcription, and SP1 activation, and emerges in fibroblasts at stage IIB as the cancer trespasses the stroma. Because scRNAseq allows a cell-type resolved analysis of these changes, we asked if the ELI signature is present specifically in fibroblasts at different stages of cancer, as well as in different fibroblast clusters. Using gene–gene correlation data within fibroblasts, which could each serve as an individual sample, we found that the ELI genes were significantly much more correlated to each other than other genes, indicating a coherent role of ELI genes in fibroblasts. Crucially, we found that resistive ELImelanoma . Again, dn genes and pro-invasive ELIup genes, respectively. We experimentally tested if genes characterizing these fibroblast clusters indeed confer the said phenotypes to fibroblasts in general. We chose to silence CTHRC1 present in cluster 5 and MMP14 present in cluster 4 in BJ fibroblasts and found that the effect of gene perturbation was consistent with the prediction. Measuring stromal invasion where fibroblasts are active players and can also be perturbed is challenging because the process is slow. We have previously developed an accelerated method to assay stromal invasability as a phenotype using a combination of nanotextured substratum, cell patterning techniques, and live fluorescence microscopy. Using this method, we experimentally determined that genes characterizing fibroblast subtypes indeed were partly contributing to the predicted property related to stromal invasability. Most of our understanding of the mechanisms driving cancer metastasis is centered on cancer cells as the primary agent, with the stromal environment considered as a passive barrier. Our framework suggests that stromal fibroblasts may be active regulators of the process of cancer metastasis itself. Resolved stage-wise analysis of fibroblasts in PDAC cancer showed a remarkable dynamic, but the most crucial for regulating cancer metastasis was an initial homogenous fibroblast reaction to contain the tumor, which collapses in cases where metastasis has set in. Two distinct fibroblast clusters, cluster 5 and cluster 4 in the current analysis, were present, respectively, in pre-metastatic stage IIA and stage IIB wherein lymph metastasis has occurred, and were enriched in pro-resistive ELIOverall, the data strongly indicate that the stromal trespassing of cancer cells to spread to proximal lymph nodes away from the primary lesion is orchestrated by a dramatic reduction in the stromal invasibility, achieved by systemic changes in gene expression in fibroblasts for anti- and pro-resistive genes. Our detailed transcriptomic exploration of fibroblasts in pancreatic cancer across early stages of metastasis presents a nuanced understanding of stromal response to cancer, and sets it in the context of the regulation of stromal resistance, or invasibility as a selected and evolved phenotype. Our work highlights that published large omics data could be harnessed to provide a deeper understanding of cancer pathogenesis, center cancer-associated fibroblasts as central players in regulating progression of a tumor into a metastatic disease, and provide a roadmap to target stromal biology to contain early cancer dissemination. Our data confirm that the complexity of stromal response to cancer is really a function of stage-wise emergence of distinct fibroblast clusters, characterized by distinct gene sets which confer initially a predominantly pro-resistive and then a pro-invasive property to the stroma.


Loeffler-endocarditis (LE) is considered a chronic restrictive cardiomyopathy and manifestation of eosinophilic myocarditis characterized by eosinophilic infiltration. LE is a rare underdiagnosed disease and associated with high morbidity and mortality.We report a case of a 46-year-old man suffering from LE associated with thromboembolic events without peripheral eosinophilia. The patient presented with typical clinical signs of acute onset of limb ischaemia, predominantly on the right limb, indicating immediate iliacal thrombectomy and due to a severe compartment syndrome additional fasciotomy. Total occlusion also of left popliteal artery suggesting an impaired chronic and aggravated impaired perfusion indicated also urgent left sided revascularization. Subsequent echocardiography revealed severe left ventricular dysfunction with a striking amount of spontaneous echo-contrast, noticeable in the left ventricular cavity. Furthermore the initial CT scan demonstrated asymptomatic left kidney- and brain infarctions. Diagnostic workup including endomyocardial biopsy (EMB) of the left ventricle, uncovered an underlying LE without peripheral eosinophilia.This case demonstrates and highlights the findings, treatment and outcome of a patient with LE and associated thrombo-embolic events without peripheral eosinophilia and emphazises the importance of awareness for LE in patients presenting with an acute cardiac decompensation and thrombo-embolic events. EMB should be performed early in unstable patients unsuitable for cardiovascular magnetic resonance imaging.The online version contains supplementary material available at 10.1186/s12872-022-02911-3. Endocarditis parietalis fibroplastica or Loeffler-endocarditis (LE) is a rare, underdiagnosed idiopathic systemic disease, first described in 1936 –5. In paA 46 years old, previously healthy man presented with typical clinical signs of acute onset of limb ischemia , predominantly on the right limb. An emergency contrast enhanced computed tomography (CT) scan of the complete aorta to thighs was performed, based on the initial suspicion of an aortic dissection. An aortic dissection however was ruled out. Main findings were a complete occlusion of the distal right common-, the right external- and internal iliac artery and common femoral artery. Furthermore, an occlusion of the left profound femoral artery and mild arteriosclerosis was found, the occluded vessel segments however were characterized by hypodense thrombotic material Fig. . The CT After the patient had gone through a SARS CoV2 infection he was finally discharged from hospital free of symptoms 8 weeks after admission. Last follow-up echocardiography revealed an unchanged LV dysfunction with an estimated ejection fraction of 34%, associated moderate LV hypertrophy and decreasing spontaneous echo contrast. Heart failure medication in combination with anticoagulation and continued immunosuppressive therapy including prednisolone and azathioprine was continued. The patient was equipted with a LifeVest® as wearable cardioverter defibrillator to protect patients at risk of sudden cardiac death.To our knowledge, this is the first report of multiple thrombo-embolic events in a patient with LE without peripheral eosinophilia. It provides new information on the natural history of this rare but clinically relevant disease.In LE cardiac involvement with migration of eosinophils into the myocardium follows a stepwise pattern from subclinical acute necrosis of the myocardium , thromboNoninvasive multimodality imaging, including transthoracic- and transesophageal echocardiography as the mainstay of diagnostic imaging and surveillance for LE could, in our case, not provide classic findings as LV wall thickness, valvulopathy or LV thrombus , 10, 18.A beneficial effect of immunosuppressive therapy with prednisone and azathioprine with significant improvement of LV function and decrease of LV dimensions was first shown in a randomized placebo-controlled study by Frustaci et al. in 2009 . In the The goal of recommended medical therapy in LE, including heart failure management and anticoagulation if presence of an intracardiac thrombus, is a decrease of eosinophil-mediated end-organ damage and prevent adverse thrombotic events , 16, 28.Decision for premature implantable cardioverter defibrillator system remain challenging in patients with impaired LV function (ejection fraction ≤ 35%), but should be avoided in patients with inflammatory cardiomyopathy as LV function may improve significantly with guideline-based heart failure therapy . Close sIn conclusion, LE without evidence of peripheral eosinophilia and absence of intracardiac thrombus formation, as demonstrated in this unique patient report, can be associated with life-threatening systemic thrombo-embolic events. Our case highlights the importance of considering LE as an important differential diagnosis in the setting of an acute cardiac syndrome in combination with thrombo-embolic events and helps to identify characteristic features found in patients with LE.early multimodality imaging including transesophageal echocardiography and if possible CMR imaging to explore not only the presence of an LV thrombus but also possible spontaneous echo-contrast to consider immediate anticoagulation aiming to prevent thrombo-embolic eventsEMB should be performed early in patients with severely impaired LV dysfunction of unknown etiologyImmunosuppressive therapy should be initiated promptly in a patient with proven LEFor strategic recommendation based on the experience in our particular case, we suggest in a patient suspicious for LE the following take-home messages:Additional file 1: Video 1. Transesophageal echocardiography showing severe systolic LV dysfunction and a striking amount of spontaneous echo contrast but no evidence of LV thrombus in a 3-chamber view (video clip).


Nanomaterial-based solutions for microorganism-related issues are gaining interest in medical fields, consumer applications, and agriculture. The main issues driving innovation in this field are the increasing antimicrobial resistance (AMR) of conventional antibiotics and the environmental toxicity of widely used agricultural chemicals—a problem that has been largely overlooked. Therefore, the synthesis of new materials or modification of the existing ones is urgently needed to tackle these health and environmental problems. The top priority of these issues was reflected by the topics of the research papers submitted to the Special Issue “Nanomaterials and Microorganisms”. Namely, the common denominator of the papers collected in this Special Issue was the design of innovative nanomaterials that showed improved activity against pathogenic bacteria. The application fields of these materials ranged from medicine to agriculture and the proposed novel materials varied from nanocomposites based on traditional antibacterial nanomaterials such as silver or zinc oxide to selenium-based or reduced graphene oxide/S/Se nanocomposites.Bacillus cereus, a bacterial strain isolated from soils of rice fields at Zhejiang University, Hangzhou, China [One of the increasingly used approaches to produce nanomaterials with efficient antibacterial properties is using a green synthesis process that involves either plant extracts or living microorganisms. During this synthesis process, which is applicable to metal-based nanomaterials, the resulting materials are rendered highly antibacterial either by a coating or structural incorporation of bioactive compounds originating from the biological materials used for synthesis. Such an approach was used by Ahmed et al. to biologically synthesize ZnO nanoparticles (NPs) in the culture supernatants of u, China . The biou, China . The pro2O3) NPs specifically interacted with the membrane of Gram-negative Escherichia coli and exerted antibacterial effects [2O3 NPs did not have antibacterial properties. Interestingly, the positive α-Fe2O3 NPs selectively acted on Gram-negative bacteria, but not on Gram-positive Staphylococcus aureus. Both the above-mentioned bacteria are clinically relevant models for the evaluation of novel antibacterial compounds. Furthermore, the NPs did not affect the bioluminescence of the naturally luminescent marine bacteria Vibrio fischeri (EC50 > 1000 mg/L) which suggests that these NPs would be safe in environmental applications.Charge-dependent antibacterial activity of NPs is a key issue that needs to be considered in the design of new nanomaterials. This topic was addressed by Vihodceva et al. who demonstrated that positively charged spherical hematite α-Fe2O NPs specOther ways to develop environmentally compatible effective antibacterial NPs include the use of natural polysaccharides for the synthesis of nanocomposites. Perfileva et al. synthesized selenium (Se) nanocomposites based on natural polysaccharide matrices arabinogalactan, starch, and kappa-carrageenan which were shown to increase potato productivity . Such st2O3 composite embedded into ethylene-co-vinyl acetate coating yielded an effective photocatalytic material which distinctly and rapidly inhibited bacterial growth whereas reactive oxygen species, and not the shedding of Ag-ions, played the main role in the photocatalytic bacterial inactivation [In environmental applications as well as consumer products, including medical settings, antibacterial surfaces are considered beneficial for eradicating pathogenic bacteria. Nanotechnology is considered an innovative approach to improving the efficiency of such surfaces. Specifically, nano-enabled photocatalytically active coatings are being developed and improved to render surfaces antibacterial. As reported by Vihodceva et al., a combination of silver and hematite NPs as a Ag/AgCl/α-Fetivation . In addiThe photocatalytic mode of action of antimicrobial nanomaterials is not always applicable in human medicine and thus different approaches must be used. For example, Nobre et al. demonstrated that hydroxyapatite which is a calcium–phosphate ceramic material with known anti-adhesive properties, functioned well as an antibiofilm material when used as a nanosuspension . The stuS. aureus and Enterococcus faecalis (growth inhibition >90% at 200 mg/L) due to membrane damage and oxidative stress.AMR-related issues were addressed in two papers of the Special Issue. Brar et al. developed nano-encapsulated drugs consisting of chitosan and silica NPs containing tetracycline and chlorpromazine . The encNanomaterials [In summary, the papers in this collection have proposed several new innovative nanomaterials with different modes of action against bacteria, thereby contributing to finding new solutions for combating the health issues of current antimicrobials. In addition to developing efficient antibacterial NPs, the impacts on the beneficial bacteria and microbiota need to be considered. The research in this direction is gaining momentum, as also illustrated by the recent publications in aterials . Combini


BackgroundOrthodontic tooth movement relies on sliding mechanics usually achieved by sliding the archwire through brackets. Sliding causes friction which is a force resisting the relative motion of two contacting objects. Frictional resistance is undesirable in orthodontic tooth movement because the archwire might bind with the bracket and prevent tooth movement. In addition, friction causes bending of the archwire leading to unwanted tooth movement or space loss through anchorage interference, prolonging the treatment time and root resorption. This study was performed to compare the frictional resistance produced by different types of ceramic brackets and stainless steel brackets with Teflon-coated stainless steel and stainless steel archwires. The surface texture of the wire before and after friction test was also evaluated using a scanning electron microscope (SEM).MethodologyA total of 48 samples were tested. In total, 12 premolar brackets each of stainless steel , monocrystalline ceramic , polycrystalline ceramic , and ceramic bracket with a metal slot  having an 0.022-inch slot were coupled with 0.019 × 0.025-inch stainless steel and Teflon-coated stainless steel wires. Each bracket-wire assembly was vertically mounted and clamped to the jaws of the universal testing machine. The wire was pulled across the bracket with a cross head speed of 10 mm per minute. The readings obtained were recorded. To evaluate the surface roughness, wires were examined using an SEM  before and after testing.ResultsUnder the testing conditions, the stainless steel bracket-stainless steel wire combination produced the least frictional resistance, and the polycrystalline ceramic bracket-stainless steel wire combination produced the highest frictional resistance. Ceramic brackets with a metal slot generated lesser friction than other types of ceramic brackets but more friction than stainless steel brackets. Moreover, for all bracket-archwire combinations, Teflon-coated wires generated reduced frictional resistance compared to stainless steel wires. The surface examination of Teflon-coated stainless steel wire and conventional uncoated stainless steel wire revealed that Teflon-coated wire had a smoother surface compared to uncoated stainless steel wire.ConclusionsWithin the limitations of this study, it was concluded that the stainless steel bracket produced the lowest frictional resistance and the polycrystalline ceramic bracket produced the highest frictional resistance. Ceramic brackets with a metal slot showed a coefficient of friction that was more than but comparable to that of stainless steel brackets. Monocrystalline ceramic brackets generated lesser friction compared to polycrystalline ceramic brackets. Further, Teflon coating of stainless steel archwires can reduce frictional resistance compared to conventional uncoated stainless steel archwires. The surface of Teflon-coated stainless steel wires was found to be smoother than uncoated stainless steel wires. Orthodontic tooth movement occurs by the application of sliding mechanics using brackets and archwires. This generates friction in the appliance which opposes every movement the orthodontist desires, impeding optimum tooth movement . In orthDuring retraction of the teeth, the archwire slides through the slots of the brackets inducing frictional forces, which, in turn, counteract forces generating tooth movement . Higher Factors affecting friction in orthodontic tooth movement can be broadly classified into mechanical and biological factors. Mechanical factors include bracket material, slot size, bracket width, bracket/archwire angulation, archwire material, archwire shape, the surface roughness of the archwire, ligature material, and force of ligation. The biological factors that affect friction include saliva, plaque, and acquired pellicle .The material with which the bracket-archwire assembly is fabricated is one of the predominant factors affecting orthodontic tooth movement using sliding mechanics. Therefore, the choice of the material is crucial to reduce the frictional forces, thereby achieving the best possible treatment outcomes. The traditional combination of stainless steel bracket and archwire outperforms any other bracket-archwire combination, with their frictional characteristics so satisfactory that they are used as the gold standard .Over time, patients’ concerns switched from esthetic appearance after the treatment to during the treatment which led to the development of smaller and more invisible bracket and archwire assemblies . The intIn 1980, the field of orthodontics witnessed the first ceramic system, composed of monocrystalline and polycrystalline ceramic, which, unlike the plastic brackets, resisted staining and could withstand heavier forces without distortion .Nevertheless, in practice, any material used will generate friction, making it impossible to develop a frictionless system. To provide the least hindrance to tooth movement it is advised to control friction rather than trying to eliminate it . This stInclusion and exclusion criteriaThe following four types of 0.022-in slot MBT prescription premolar brackets were tested: monocrystalline alumina, polycrystalline alumina, polycrystalline ceramic bracket with stainless steel slot, and stainless steel bracket. The Different types of brackets used were standard twin brackets with four tie wings for the use of elastomeric ligatures. The ceramic brackets are made up of aluminum oxide. A total of 12 brackets of each type were used . A total of 48 bracket-wire samples were studied. Each bracket was tested only once, and each wire specimen was drawn through one bracket only to eliminate the influence of wear. Different bracket-wire combinations were divided into different groups Table -8.A stainless steel bar measuring 100 × 15 × 2 mm was used for mounting the bracket-archwire assembly. A line measuring 1 mm in depth was inscribed in the midline, parallel to the long axis, to act as a guide for reproducible bracket positioning. This was done to help align the pull of the wire through the bracket slot so that friction is not induced by adverse tipping or torsion moments. The different types of ceramic brackets and stainless steel brackets were bonded onto the stainless steel bar with cyanoacrylate gum using a bracket placer and 0.021 × 0.025-inch stainless steel jig.Stainless steel  and Teflon-coated stainless steel archwire  segments were then ligated to the brackets with the help of elastomeric modules . One end of the stainless steel bar was attached to the universal testing machine using clamps and the other end was suspended free Figure . The arcThe surface roughness of the stainless steel and Teflon-coated stainless steel wires were examined before and after testing for friction using an SEM. The stainless steel and Teflon-coated stainless steel wires were cut to 10 mm in length. As Teflon-coated stainless steel wire is nonconductive, gold sputtering had to be done before examining surface characteristics using SEM Figure . After tStatistical analysisThe data were tabulated in Microsoft Excel 2010 and statistical analysis was performed. Descriptive statistics were presented as mean, standard deviation (SD), minimum, and maximum values. The values were tested for normality using the Shapiro-Wilk test. Because the data were not normally distributed, The Kruskal-Wallis test with post hoc test was performed for inferential statistics. P-values less than 0.05 were considered statistically significant.Friction measurement using the Kruskal-Wallis test showed a significant bracket effect (p < 0.001). Post hoc pairwise comparisons showed that the polycrystalline ceramic bracket had the highest frictional force, which was statistically significant, followed in decreasing order by monocrystalline ceramic bracket, ceramic bracket with a metal slot, and stainless steel bracket. The Kruskal-Wallis test showed a significant wire alloy effect (p < 0.001). Post hoc pairwise comparisons showed that the Teflon-coated stainless steel archwire produced frictional resistance comparable to the stainless steel archwire Table .SEM examination of the archwire surfaces at 250×, 500×, 1,000×, and 5,000× magnifications showed a smoother surface of the Teflon-coated stainless steel archwire when compared to conventional stainless steel archwire before the friction test. Archwire roughness and frictional resistance showed a positive correlation. SEM examination after the friction test revealed an increase in the surface roughness of Teflon-coated stainless steel archwire compared to stainless steel archwire Figures -15.Most kinds of orthodontic treatment often have some form of sliding mechanics to move the teeth. Sliding mechanics can be used to align irregular teeth or close extraction spaces. Friction is inadvertently present between the bracket-wire assembly, thereby reducing the efficiency of orthodontic treatment. Therefore, frictional forces must be overcome to produce tooth movement during alignment and space closure. Frictional forces arise between two surfaces in contact when there is relative motion between the two, or the potential for it, and when the contacting surfaces are not perfectly smooth. The magnitude and variability of the frictional forces determine the efficiency of treatment and the reproducibility of results when similar activation forces are applied. Efficient orthodontic tooth movement requires the application of light continuous forces. To apply light forces, the selection of materials is important. Orthodontists can choose from an extensive inventory of materials such as metal, ceramic, polymer, and composite. Each archwire-bracket combination has a unique set of physical properties that may make it perform differently from others .The current study was undertaken to evaluate the static friction of stainless steel and Teflon-coated stainless steel wires measuring 0.019 × 0.025 inches combined with stainless steel, polycrystalline ceramic, polycrystalline ceramic bracket with a metal slot, and monocrystalline ceramic bracket (0.022 × 0.028 inches). A sample size of 48 was calculated using statistical analysis, which was divided into eight groups based on the bracket-wire combination. Each test specimen was tested for frictional resistance in the universal testing machine, and the same test specimen was evaluated under an SEM to check for surface roughness before and after the friction test.The results of this study show that there is a significant difference in frictional resistance when different combinations of brackets and archwires are used. Thus, the null hypothesis that frictional resistance is not affected by the different bracket and archwire materials was rejected. The mean frictional resistance ranges from 0.81 to 3.13 N. The highest frictional resistance was displayed by polycrystalline bracket and stainless steel wire combination (3.13 N), and the lowest frictional resistance was displayed by stainless steel bracket and stainless steel wire combination (0.81 N). For ceramic brackets used in this study, both polycrystalline and monocrystalline brackets in combination with stainless steel archwire showed high frictional resistance compared to conventional stainless steel bracket and stainless steel archwire combination. This is in accordance with the study conducted by Fidalgo et al. on friction between the different wire and bracket combinations in artificial saliva. The authors reported that there was a significant difference in friction between ceramic bracket-stainless steel archwire combination and stainless steel bracket-stainless steel archwire combination . PrattenIn this study, two groups contained polycrystalline ceramic brackets with a metal slot in combination with stainless steel wire and Teflon-coated stainless steel wire. Both the groups have shown superior results compared to polycrystalline and monocrystalline ceramic bracket groups by displaying reduced frictional resistance. These results are supported by the study conducted by Chaa et al. on the friction of conventional and silica-insert ceramic brackets in various bracket-wire combinations. They concluded that the polycrystalline alumina bracket with a metal slot showed significantly lower friction than ceramic brackets .Two types of archwires, stainless steel, and Teflon-coated stainless steel wire were used in this study in combination with different types of brackets. The results of this study show that the wire-bracket combinations with Teflon-coated stainless steel archwire had significantly less frictional resistance when compared to stainless steel wire. These results correspond with the study conducted by Farronato et al. They concluded that, for all bracket-archwire combinations, Teflon-coated archwires resulted in lower friction than the corresponding uncoated archwires, and the results showed that Teflon coating has the potential to reduce resistance to the sliding of orthodontic archwires .Another important characteristic of orthodontic wires is their surface roughness. It affects various factors such as friction, the amount of microbial plaque accumulation, and corrosion of wires, which, in turn, plays a role in orthodontic treatment . Teflon The results of this study showed that Teflon-coated stainless steel wires show lesser surface roughness compared to stainless steel wires, which is in accordance with a study conducted by Mousavi et al. to evaluate the effect of esthetic coating on the surface roughness of orthodontic archwires. They compared the surface roughness of four coated esthetic wires with that of a conventional orthodontic wire and concluded that coated wires have significantly less frictional resistance compared to conventional uncoated wires . Rudge eIn this study, the post-frictional test SEM analysis of Teflon-coated archwire showed increased surface irregularities compared to that of stainless steel archwire. Therefore, the Teflon-coated stainless steel wire likely generates higher frictional resistance than stainless steel wire when used for a longer duration. This increase in frictional resistance can be attributed to the increase in surface roughness due to distortion of the coating during the sliding of the archwire through the bracket .Therefore, considering all the above-mentioned findings, the stainless steel bracket-stainless steel wire combination generated the least frictional resistance, and the polycrystalline ceramic bracket-stainless steel wire generated the highest frictional resistance. The surface roughness of Teflon-coated wires was found to be smoother than the conventional uncoated stainless steel wire.The limitations of this study are that the influence of saliva, temperature, humidity, changes in the ligation force, inter-bracket distance, and movements occurring during mastication were not considered. All these factors cause variations in the frictional values. Nevertheless, the results obtained in this in vitro study can be carefully generalized to clinical conditions as a useful guide for the selection of bracket-archwire combinations to obtain good sliding mechanics in orthodontic applications.The stainless steel bracket and stainless steel archwire combination generated the least frictional resistance. The stainless steel bracket with Teflon-coated stainless steel wire combination produced the second lowest frictional resistance and was comparable with that of the stainless steel bracket and stainless steel wire combination. Therefore, Teflon-coated stainless steel wires can be used as an alternative to conventional stainless steel wire during retraction using stainless steel brackets as they are more esthetic. All types of ceramic brackets produced higher frictional resistance compared to stainless steel brackets. Ceramic brackets with a metal slot generated lower friction with both stainless steel and Teflon-coated wire, which was comparable to that produced by the stainless steel bracket-stainless steel archwire combination.The surface characteristics of Teflon-coated stainless steel wire were smoother compared to stainless steel archwire before the friction test. The surface of Teflon-coated stainless steel wire was more irregular compared to conventional stainless steel when observed using SEM after the friction test. Therefore, friction may increase following the use of Teflon-coated stainless steel wire for long durations.


The global coronavirus outbreak was viewed as a severe threat to healthcare providers, particularly nurses. COVID-19 has numerous public health management dimensions, including the reorganization of health care workers to support and assist patients.This study used a qualitative approach to gain an insight into the experiences of psychiatric nurses who were treating quarantined patients at various hospitals. This research aimed to investigate the experiences of reassigned psychiatric nurses during the COVID-19 outbreak in Hungary.Using a phenomenological approach, we enrolled 7 nurses who provided care for COVID-19 patients from July 2020 to April 2021. The interviews were conducted face-to-face in the form of semi-structured interviews and were analysed using a health-psychology approach: interpretive phenomenology analysis.Our study shows that pandemic public health reorganization creates novel situations and issues that nurses are forced to address. Our findings suggest that three themes emerge from the data to describe psychiatric nursing: (1) Usage of earlier clinical experiences, (2) Recognizing mental issues, (3) Social networks.This study suggests professional self-concepts and job satisfaction in relation to treating quarantine patients are affected by the identity and conflicts of psychiatric nursing in a novel situation.No significant relationships.


The zero-gap design ensured efficient intermittent operationfor a week, while avoiding significant performance loss.We demonstrate thedynamic operation of CO Electrochemical power-to-gas and power-to-liquidtechnologies are promising chemical energy conversion approaches tobe coupled with intermittent renewable energy sources to balance thegrid while utilizing cheap excess energy at peak times.2 Electrochemical hydrogen generation methods (suchas proton exchange membrane (PEM) water electrolyzers) are well knownfor their dynamic response to external electrical power load; therefore,they can be directly coupled to different renewable energy sources,such as windmills or solar photovoltaic (PV) power plants.In the future electrical energygrid, an increasing fluctuation of the power load is expected becauseof the growing amount of intermittent renewable energy in the electricitymix.2 electrolyzers operating at industrially relevantcurrent densities5 can, in principle, offer similar opportunities, althoughno experimental evidence has been demonstrated on this matter yet.Such verification would be very important, considering the notabledifferences compared to water electrolyzers , etc.). Specifically, fluctuations in the local pressure, dueto the rapid increase/decrease in the reaction rate, might cause floodingin the cathode GDE, which can be detrimental for the stability ofthe electrolyzer cell. A few studies targeted dynamic operation, butthey were limited to on/off switching cycles in short measurementsand some simple variation of the potential/current to regenerate theCu catalyst and/or avoid precipitate formation in the electrolyzercells.10 Low-temperature CO2 electrolyzers are particularly promisingfor dynamic operation, while high-temperature systems are challengedby their thermal management .11 In this Energy Express, we compare the dynamicoperation of two low-temperature CO2 electrolyzer cells:a membrane-less microfluidic and a zero-gap cell, to provide the first experimental demonstration of an electrolyzercell absorbing a power load mimicking a PV power plant, generatingCO for a whole week.DirectCOFigure S1) for the autonomous testing of CO2 electrolyzercells  in the CO2-to-CO conversion process .As the first step, we defined a dynamic current control protocol totest the response of zero-gap electrolyzer cells in terms of cellvoltage and CO/H2 partial current densities (jCO and j2H). Theprocess consisted of alternating 8 h periods of constant current anddynamically changing current operation . We decided to control the current density of the cellbecause proper power electronics are available for both DC/AC/DC andDC/DC conversions, as demonstrated on the example of PEM water electrolyzers.12 Comparison with constant current operation  shows that the stability  is not affected by the dynamicallychanging current pattern. In fact, one may even consider some possiblebeneficial effect of the current fluctuation, such as the releaseof entrapped gas bubbles.First, we have developed an environment, and ourcurrent control protocol enhances the fluctuations in the power . As shownin jCO follows the shape of the total current curve(FE2H remains below 15%). The cell voltage curvealso resembles that of the power input . To assess the long-term stabilityof the cell under these conditions, we ran the electrolyzer for aweek. As seen in Subsequently, we applied a protocolwhich mimics the power outputof a solar PV power plant in Germany, where data was available with1 min resolution.Figure S8 illustrates stable operation at fixed current density, but the cellgradually gets flooded once the current density is varied. The rapidlychanging gas evolution rate can induce rapid fluctuation in the local pressure. As the gas/solutioncan penetrate into the carbon paper even at low differential pressures,15 it is not surprising that the GDE gets flooded.To check whether this capability is a unique feature of zero-gapcells or can be stretched to microfluidic devices, we extended ourstudies to this latter group. Overall, wedemonstrated that the studied zero-gap cell can properlyfunction under intermittent operational conditions, while its microfluidiccounterpart suffers from rapid flooding under such circumstances.Further efforts are ongoing to elucidate the effect of dynamic powerload on multicell stacks and multistack systems (together with uncoveringfailure mechanisms), because these insights are very important indesigning electrochemical cell configurations and energy conversionsystems to scale-up this promising technology.


Head and neck squamous cell carcinoma (HNSCC) remain a substantial burden to global health. Cell-free circulating tumour DNA (ctDNA) is an emerging biomarker but has not been studied sufficiently in HNSCC.TM, a highly sensitive personalised assay using deep sequencing for tumour-specific variants, to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence.We conducted a single-centre prospective cohort study to investigate ctDNA in patients with p16-negative HNSCC who received curative-intent primary surgical treatment. Whole-exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumour tissue. We utilised RaDaRIn 17 patients analysed, personalised panels were designed to detect 34 to 52 somatic variants. Data show ctDNA detection in baseline samples taken prior to surgery in 17 of 17 patients. In post-surgery samples, ctDNA could be detected at levels as low as 0.0006% variant allele frequency. In all cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days.This study illustrates the potential of ctDNA as a biomarker for detecting minimal residual disease and recurrence in HNSCC and demonstrates the feasibility of personalised ctDNA assays for the detection of disease prior to clinical recurrence. Despite improvements in treatments for squamous cell carcinoma of the head and neck (HNSCC), many patients develop disease recurrence . Fewer tThe detection of circulating cell-free tumour DNA (ctDNA) as a marker of minimal residual disease following curative-intent surgery holds promise for identifying patients at an increased risk of relapse, who may benefit from adjuvant radio(chemo)therapy or facilitate close monitoring with repeat resection, if needed. The main advantage of ctDNA is its availability from liquid biopsies that are minimally invasive and easily obtainable throughout the course of the disease, including before any other diagnostic and/or therapeutic measures. Together with the increased cost-effectiveness of next-generation sequencing (NGS), even the smallest amounts of ctDNA can potentially be detected and quantified –8. Over TM, Inivata Ltd, Cambridge, UK) to detect ctDNA pre- and post-operatively. Here we demonstrate subsequent detection and longitudinal monitoring of ctDNA following surgery, including detection of molecular relapse ahead of clinical relapse in five of five patients who progressed, and in none of the patients without clinical recurrence.For this study, we prospectively collected plasma samples from 17 patients with p16-negative HNSCC primarily treated with curative-intent surgery. The aims of this study were firstly to determine whether post-operative ctDNA detection can act as a biomarker for surgical tumour clearance. Secondly, we wanted to evaluate the potential of personalised ctDNA analysis for early molecular-level detection of relapse prior to clinically confirmed recurrence with concomitant implications for therapy planning. We used a highly sensitive personalised assay design (RaDaRTM) in plasma of patients with HNSCC.LIONESS is a single-centre non-interventional prospective experimental evidence-generating cohort study. Patients with HNSCC of the oral cavity, pharynx or larynx with Stages III-IVb (AJCC 8th edition), p16-negative, deemed resectable and scheduled for surgery were considered eligible. Patients with distant metastasis (cM1) or other active malignancies at the time of enrolment were excluded. Seventeen patients were recruited at the Department of Otorhinolaryngology, Head and Neck Surgery at the Hospital of the Ludwig-Maximilians-University of Munich between April 2020 and April 2021, with a median follow-up of 371 days (292–532 days). As suggested by the local multidisciplinary tumour board, patients received adjuvant radio(chemo)therapy according to the National Comprehensive Cancer Network guidelines (NCCN Guidelines) , if neceg for 10 min and second centrifugation of plasma aliquots at 20,000 × g for 10 min. The buffy coat layer was separated during blood processing and stored at −80 °C. DNA was extracted from 200 µl of buffy coat (leukocytes) using QIAamp DNA Blood Mini Kit (Qiagen) and circulating cell-free DNA (cfDNA) extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Buffy coat and cfDNA were quantified using digital PCR (BioRad QX200) as described previously [Serial plasma samples were collected from 17 patients 1–4 days pre-operatively as well as 2–7 days post-operatively. Additional blood samples were obtained prior to and following adjuvant therapy, if indicated, as well as during follow-up visits. In case of a resectable recurrence, samples were obtained again pre- and post-operatively as mentioned above. Venous blood from study participants was collected by standard phlebotomy techniques in two cell-free DNA blood collection tubes  and sent for further processing to Inivata Ltd . Plasma samples were prepared from up to 10 ml of venous blood, as soon as possible after collection and within 7 days, with an initial centrifugation at 1600 × eviously .FFPE tumour blocks of the resected specimen were obtained and ten 10-µm unstained slides and one haematoxylin and eosin-stained slide were cut from representative FFPE blocks. An experienced head and neck pathologist assessed tumour content and cellularity and suitable tumour areas were marked for macrodissection, if necessary. Tumour tissue slides were sent for whole-exome sequencing (WES) to Inivata Ltd. DNA was extracted from the provided FFPE tumour tissue samples using the Maxwell RSC DNA FFPE kits. The extracted DNA was processed with the KAPA Hyper Prep Kit and indexed uniquely. The resulting pre-capture libraries were quantified using the Quant-iT dsDNA High Sensitivity assay. Each library proceeded to exome enrichment and was analysed on a fragment analyser and quantified using the Quant-iT dsDNA High Sensitivity assay. Sequencing was performed on the HiSeq4000 platform (Illumina).The analysis of tumour-only exome-sequencing data was performed using a proprietary pipeline: samples with a median cellularity of 50%  were processed through fastq files processing, alignment, and variant calling. Germline variants were filtered out using custom filters that take into account prior knowledge available from public single nucleotide polymorphism (SNP) databases and variants were filtered based on multiple parameters including allele frequency and depth. Performance characteristics of the WES is shown in Supplementary Table TM is a personalised ctDNA assay built on the InVision® platform [TM panel design.RaDaRplatform , which uTM assays were performed on cfDNA from plasma aliquots alongside a buffy coat DNA control sample, which was used for identification and removal of germline variants, the removal of variants due to clonal haematopoiesis of indeterminate potential (CHIP) from the analysis, and as a positive amplification control. Multiplex PCR was performed with input concentrations ranging from 1452 to 20,000 amplifiable copies per sample, as measured by droplet digital PCR, with a median value of 14,550 copies.Following primer panel qualification, RaDaRTM libraries were sequenced using the Nova-Seq 6000 system  and sequencing data analysed in a multi-step process: fastq files were demultiplexed using bcl2fastq, reads were then aligned using the bwa mem alignment software and processed using proprietary software to identify primer pairs and count mutant and reference bases.RaDaRIndividual variants were subject to quality control in the process of calling residual disease positive or negative. Variants present in the buffy coat material or absent in the tumour tissue DNA were excluded from further analysis. Proprietary methods were used to call residual and recurrent disease: a statistical model was used to assess the statistical significance of the observed mutant counts for each variant and the information was integrated over the entire set of personalised variants to obtain evidence of tumour presence or absence at the sample level. This includes an assessment of the noise of each individual variant class and the sensitivity and specificity based on the number of variants in the panel. A sample will be called as positive for residual disease if its cumulative statistical score is above a pre-set threshold, as defined during analytical development . The tumA time course of ctDNA detection levels was plotted from the date of pre-operative sample collection. Red dots indicate ctDNA-positive samples, and black dots ctDNA negative samples. Progression is indicated with a yellow triangle, based on annotated clinical recurrence. ctDNA levels are estimated as part of the calling procedure. ctDNA negative samples were assigned a VAF of not detected (ND) and plotted at the bottom of the plot. Lead time was taken to be the interval between detection of the first ctDNA-positive sample post surgery and confirmation of clinical recurrence.Survival analysis was plotted using a Kaplan Meier Curve, although the limited sample number only allowed qualitative assessment. Each curve represents a specific cohort stratified according to specific criteria—ctDNA detection yes/no—and should approach the true survival function for the population under investigation, provided the sample size is large enough. Each vertical drop in the curve represents an event occurring, in this case relapse: for instance, for the ctDNA-positive patients (red curve), each drop identifies one patient recurring as detected by clinical investigations.Seventeen patients with resectable disease at the time of diagnosis were recruited into the LIONESS study and underwent longitudinal blood sampling. 103 serial plasma samples were assessed for evidence of ctDNA  on a background of a previous pT2 pN0 floor of the mouth SCC that had been treated surgically and with adjuvant radiotherapy more than a decade ago. Local resection of both tumours was performed with curative intent. The defect was reconstructed with a supraclavicular artery island flap (SCAIF) and bilateral neck dissection was performed. The patient underwent additional surgery a few days later due to positive resection margins in the floor of the mouth as well as developing a fistula, which was treated with another SCAIF. No adjuvant treatment for Stage IVa disease was possible because of radiation doses the patient had received in the past. Personalised panels were designed for each tumour entity, with 47 somatic variants passing all quality control filters for the laryngeal tumour and 35 somatic variants for the floor of the mouth tumour , as confirmed by intra-operative frozen section analysis. Once again, the rise in ctDNA levels preceded clinically confirmed local relapse 110 days later Fig. . Due to Patient 9 had a pT3 pN0 tumour of the lateral tongue, which was resected in addition to removing the cervical lymph nodes. Only ten somatic variants passed all quality control filters for the personalised ctDNA assay due to a high number of germline variants or variants that were absent in the primary tumour DNA at panel quality control. Pre-operative ctDNA was detected at 0.257% VAF and decreased immediately after surgery to undetectable levels Fig. . Prior tFinally, patient 2 exhibited a similar pattern of minimal residual disease and rising ctDNA levels ahead of clinical recurrence Fig. . FollowiPatient 6 had a pT3 pN0 (Stage III) laryngeal cancer and underwent laryngectomy and bilateral neck dissection with curative intent. Of the variants included in the personalised ctDNA assay design, 46 somatic variants were selected. Pre-operatively, ctDNA was detected at 0.336% VAF and decreased immediately after surgery to undetectable levels Fig. . They rectDNA was detected for all patients who had a clinical recurrence, as described above and shown in Supplementary Fig. TM, a highly sensitive personalised assay using deep sequencing of up to 48 primer pairs each capturing at least one tumour-specific variant to analyse serial pre- and post-operative plasma samples for evidence of minimal residual disease and recurrence. Detection of ctDNA was shown to be sensitive to levels of 0.0006%, consistent with previous studies utilising RaDaRTM in breast [This study prospectively evaluated the performance of ctDNA monitoring in a cohort of 17 patients with p16-negative HNSCC planned for curative surgery. We utilised RaDaRn breast  and lungn breast .TM assay, we were able to detect pre-operative ctDNA in 100% of patients. In all five cases with clinical recurrence to date, ctDNA was detected prior to progression, with lead times ranging from 108 to 253 days.Previous studies have shown that ctDNA could be measured in the blood of 70% of patients with HNSCC Stage I and II, in 92% with Stages III and IV  and in ~In five case studies presented here, we demonstrate how ctDNA detection could have been applied to aid therapy planning and clinical decision-making. Depending on the disease stage, reconsideration for proceeding with close clinical monitoring may even be possible, particularly for those patients where the disease was discovered early enough and where adjuvant treatment may be considered optional. Confirmation of post-operative tumour clearance by undetectable ctDNA could potentially allow a more flexible start of additional therapeutic measures. However, interventional studies are needed to evaluate whether ctDNA monitoring could be an additional tool to stratify patients for adjuvant therapy or clinical follow-up.In contrast to suggested tumour clearance by undetectable ctDNA levels, positive post-operative ctDNA or an increase of ctDNA levels soon after tumour resection may indicate minimal residual disease independently from intra-operative analysis of frozen sections. With this knowledge at hand, the operating surgeon may consider additional resection(s) of the tumour bed or revision neck dissection, particularly in cases where adjuvant treatment will not be possible or plan further surgeries accordingly. In cases where additional operative procedures are not feasible, a timely start of adjuvant treatment should be expedited. It is noteworthy, that when patients decline adjuvant therapy, as has been the case with patient 13, results from a personalised ctDNA assay may offer additional means to persuade an indecisive patient to undergo necessary and potentially life-saving adjuvant radio(chemo)therapy. Having a single blood test done, even at a local GP practice, may also improve compliance with clinical follow-up appointments in this patient cohort where addictive behaviour, including alcohol abuse and smoking, as well as lack of adherence to medical recommendations is frequently experienced –53. NeveTM was able to detect ctDNA post-operatively in five cases, preceding the clinical diagnosis of recurrence by many weeks. In most, if not all cases, earlier detection of recurrence may lead to a less radical choice of therapy and may even be the only chance of cure for the patient.Furthermore, rising ctDNA levels prior to start of adjuvant treatment may trigger re-assessment of distant lesions initially deemed non-suspicious in pre-operative imaging. CT imaging of patient 2 done pre-operatively, for example, showed several, very small subpleural lesions in the lung, that, retrospectively, could well correspond to the subsequently confirmed disseminated pleural metastases. Differentiating a single pulmonary metastasis from primary lung SCC is of high clinical importance, but not possible in most cases with current diagnostics because these tumours often share the same histomorphology and immunohistochemical profiles . This knCT scans post-treatment as part of the routine clinical follow-up are commonly performed, helping clinicians to make choices about further clinical management. Here, the evaluation of ambiguous radiological findings may also be aided by personalised ctDNA analysis. For instance in patient 9, a CT scan conducted as a result of a clinically palpable submucosal induration of the tongue was reported to show no sign of local recurrence. Importantly, panendoscopy and biopsy subsequently confirmed the clinical suspicion of local relapse. In retrospect, ctDNA detection at the time of a clinically palpable tongue induration clearly indicated recurrence. Importantly, the MRI scan conducted after histopathological confirmation of a recurrence only showed a diffuse contrast enhancement within the left intrinsic musculature of the tongue even though the tumour was found to be more than 3 cm in diameter (yrpT3) following resection. This observation may suggest that ctDNA analysis offers a unique opportunity to alter patient management, therefore potentially improving survival. In addition, it may also spare patients from unnecessary and risky endoscopic procedures triggered by inconclusive findings of CT and/or PET-CT scans if ctDNA remains undetectable during follow-up. However, further studies are needed to support this.Interestingly, there is only limited evidence for the effectiveness of follow-up in patients with HNSCC. No international consensus concerning the intervals and duration for follow-up examinations exists, or details about techniques to be used . Currentn = 17) enrolled to date as well as the still ongoing follow-up with the shortest follow-up length amounting to 10 months. We acknowledge that more patients of this cohort are likely to recur at a later stage, however, using RaDaRTM we were able to detect early molecular-level recurrence within the first 6 months of follow-up. Larger cohort sizes with longer follow-up are needed to allow for sufficiently powered statistical analyses and to correlate ctDNA detection with recurrence and survival. Furthermore, ctDNA detection is reported at the sample level, giving a high degree of sensitivity, but does not inform on the levels and tracking of individual variants or the emergence of sub-clones. In addition, our personalised ctDNA assay design requires suitable FFPE tumour material to be available for WES, which may not always be achievable and may limit the use of this method in certain specific cases. However, a strength of our study includes the use of a personalised ctDNA assay design that is highly sensitive and specific and can be applied to all patients with available WES data. Analytical validation demonstrated that RaDaRTM’s sensitivity approximately doubles each time the number of variants tracked double (TM assay comparable to other platforms for similar applications, including large fixed NGS panels that are in widespread use.Limitations of this study include the relatively small number of patients (d double . The majIn summary, findings from this prospective cohort study illustrate the potential of ctDNA as a biomarker of minimal residual disease detection and monitoring of early molecular-level recurrence in patients with HNSCC, demonstrating the feasibility of personalised ctDNA assays for therapy planning and follow-up. In this cohort, ctDNA was detected prior to surgery in 17 of 17 patients with HNSCC and during follow-up in all five patients who subsequently relapsed, with lead times ahead of clinical recurrence ranging from 108 to 253 days. In addition, although ctDNA levels will be displaying varying dynamics throughout the course of a patient’s management, personalised ctDNA monitoring could also be offered to patients primarily treated with non-surgical modalities. Early detection of relapse using ctDNA could therefore indicate patient populations where earlier therapeutic intervention may be beneficial. However, further interventional prospective studies with sufficient power are required before ctDNA analysis can be introduced into routine daily clinical practice.Supplementary legendsSupp MethodsSuppl Figure 1Supp Figure 2Supp Table 1Supp Table 2Supp Table 3