The separation of discrete fossiliferous levels within an archaeological or paleontological site with no clear stratigraphic horizons has historically been carried out using qualitative approaches, relying on two-dimensional transversal and longitudinal projection planes. Analyses of this type, however, can often be conditioned by subjectivity based on the perspective of the analyst. This study presents a novel use of Machine Learning algorithms for pattern recognition techniques in the automated separation and identification of fossiliferous levels. This approach can be divided into three main steps including: (1) unsupervised Machine Learning for density based clustering (2) expert-in-the-loop Collaborative Intelligence Learning for the integration of geological data followed by (3) supervised learning for the final fine-tuning of fossiliferous level models. For evaluation of these techniques, this method was tested in two Late Miocene sites of the Batallones Butte paleontological complex . Here we show Machine Learning analyses to be a valuable tool for the processing of spatial data in an efficient and quantitative manner, successfully identifying the presence of discrete fossiliferous levels in both Batallones-3 and Batallones-10. Three discrete fossiliferous levels have been identified in Batallones-3, whereas another three have been differentiated in Batallones-10. The Batallones Butte, located 30 km to the south of Madrid Spain,  and 1 kmThe Batallones Butte sites have a late Vallesian age , based on the faunal association . HoweverThe decimeter to centimeter-scale separation of discrete fossiliferous levels within the same lithostratigraphic unit is common in Cenozoic sites , but tenPalaeoecological, palaeoenvironmental and taphonomical studies carried out at Batallones Butte , 2016 haThe pioneering introduction of Artificially Intelligent Algorithms (AIAs) in fields of archaeology and paleoanthropology has revolutionized numerous sub-disciplines such as those related with genetic sequencing , site anAIAs, including those trained through Machine Learning and Deep Learning (ML & DL) techniques, present numerous possibilities for the processing of highly complex and noisy data sets . CombineThe main goal of this study is to establish whether or not ML analysis can be used to quantitatively identify discrete fossiliferous levels in paleontological and archaeological sites based on the study of the spatial distribution of fossils. Specifically, this method has been applied and tested in two Batallones Butte sites  in order to determine if the deposits are homogeneous or heterogeneous with discrete fossiliferous levels.x, y and z coordinates according to their position within a grid, as well as the trend and plunge of elongated fossil remains. Additional data, for future taphonomical studies, were also collected concerning the degree of articulation between anatomical elements, the overall preservation of the remains and the restoration techniques used.The site of Batallones-3 has undergone systematic field excavations between the years 2001 and 2017, whereas excavations at Batallones-10 started in 2007 and are ongoing. Batallones-3 corresponds to the carnivoran-rich lower part of the hour-glass structure previously mentioned. It is not clear whether an upper part ever existed for this fossil site, but, if it existed, it was destroyed by slope-erosion processes . In turn, Batallones-10 corresponds to the herbivore-rich upper part of the hour-glass structure and, if present, will have a lower part, yet to be excavated. At both sites, standard fossil vertebrate excavation protocols were followed in the extraction of paleontological remains . On-sitex, y and z coordinates for in-depth 3D analysis. Manageable slices were extracted, similar to the approach proposed by x and one across the y axis, searching for representative areas with the greatest object densities to capture a global vision of the entire site. In the case of Batallones-3, located in a domically-shaped cave with a debris cone in the middle under an inferred opening, two 2-m-wide slices were obtained along the site’s x axis, one on each side of the debris cone.For the purpose of this study, the data used for both supervised and unsupervised ML applications consisted solely in the spatial distribution of remains, including their www.rproject.org, Once slices were selected, the coordinates of each of the remains were imported into the free R software, x64 v.3.5.1  algorithm from the “fpc” R package was used for pattern recognition in 3D spatial data. Proposed as a means of overcoming clustering issues where groupings of samples are not straightforward , DBSCAN DBSCAN performs through establishing areas of significant point densities, thus providing an efficient means of finding non-parametric patterns among noisy data sets while easily detecting outliers in low-density regions. This is especially useful when used for pattern recognition in spatial studies, where traditional clustering models such as partitioning and hierarchical algorithms tend to focus groupings around a centroid or subsamples in a convex manner. This frequently creates conflict in real-life circumstances.core points) from those on the border of the cluster . The differentiation between the two is established mathematically depending on the reachability of neighboring points (q) from a core point (p). To define this, DBSCAN requires two main hyperparameters, the ε value defining the neighborhood (a.k.a. Eps) of a point and the minimum number of points (MinPts) that are required to form the cluster . From here DBSCAN defines the neighborhood of p within dataset D as NEps(p) = {q ∈ D | dist ≤ ε}, adjusting for border points and the exclusion of noise through p ∈ NEps(q) and |NEps(q)| ≥ MinPts | ≥ MinPts. The final components that define point density for clustering are established through density-connected points, whereby a point o is density-reachable from both p and q, essentially connecting both border and cluster points.DBSCAN has proven efficient when applied to 2D, 3D and/or higher dimensional feature spaces . The alg≥ MinPts . The defC requires the fulfillment of conditions; (1) ∀ p, q: if p ∈ C and q fulfill the definition of being density reachable, then q ∈ C, while (2) ∀ p, q ∈ C: p must be density-connected to q. These defined conditions are named Maximality and Connectivity, respectively  and slices with a lower number of remains (MinPts ≈ 3). This abides by general rules recommended by numerous authors , who defneighbor , employineighbor . The useConsidering the currently unquantifiable nature of numerous geological features that ML algorithms may be unable to detect, a human-in-the-loop collaborative strategy was proposed for this study . The objThe Expert-in-the-Loop (EitL) approach adopted here consisted in the revision of the clustered dataset by considering possible underlying geological components that could be generating noise undetectable by the model. Components of this nature could easily include disconformities, geological faults, erosive surfaces, uneven sedimentation or even sedimentation of geological elements such as large boulders that could be separating point-densities that would essentially belong to the same layer.Machine Teaching (MT) in as much as the expert knowledge is used for troubleshooting and debugging (sense-making approach). Moreover, the amount of human input for models was monitored in a collective manner, using numerous domain experts to ensure accuracy (The strategies employed consequently used EitL (in this case geologist-in-the-loop) interaction to manually correct clustered patterns, assessing which of the clustered groups were geologically separated and assigning these clusters to a new labeled layer. This was performed as objectively as possible through complimenting paleontological data routinely collected from the site during excavation and evaluating the nature of each of DBSCAN’s groupings. EitL thus benefits through accuracy . In caseOnce spatial data points had been passed through the DBSCAN algorithm for initial pattern detection and then corrected by geologist-in-the-loop interventions, supervised algorithms were employed to define the final fossiliferous level models and classify those points in cluster group “0”.train:test] splits using k-fold cross-validation (k = 10) in order to ensure the model could efficiently adjust its weights. While some authors propose the use of Spatial Cross-Validation (SCV) for studies regarding geographic data . SVMs map out input vectors into a non-linear high dimensional feature space, using hyperplanes to calculate the degree of separation between samples (kernel function is used to define the feature space ( samples . In ordere space . The conre space . The conRandom Forest (RF). RF can be described as a robust and highly complex form of decision tree which has proven useful in the past for the processing of spatio-temporal data  and predicted (pi) values for each classification. MSE values are further interpreted considering AIAs of this type are trained to reduce the error produced in CV sets, therefore the smaller the MSE value the more powerful the predictive model. Model evaluation was performed using the “caret” R package.Model evaluations were performed following standardized ML protocol, evaluating the Kappa (κ), Sensitivity, Specificity and Balanced Accuracy values obtained through confusion matrices . SensitiOnce the optimal models were defined, these were used to classify any of the points separated as noise by DBSCAN as well as the clusters that the EitL participants could not objectively define as a part of either fossiliferous level (so as to avoid subjectivity). In order to empirically class any point as a member of a fossiliferous level, the trained SVM and RF algorithms were used to predict the class label of each point. A threshold of 80% security was established as acceptable. Any points that could not be classed by the model with over an 80% predictive decision boundary were thus rejected and therefore not classified into any level in the final fossiliferous levels models.DBSCAN clustering proved highly efficient at quickly processing each slice, grouping fossil remains into abundant clusters in both Batallones-3  and Batax axis slice (n = 141) and Batallones-3 right slice (n = 56). For Batallones-3 right slice, this can be attributed to certain areas of the profile having little separation between the layers  as noise in Batallones-10 and 62 (7.76%) in Batallones-3, with the highest levels of noise appearing in Batallones-10 e layers . Neverthx axis slice  of the debris cone . The lefBatallones-10 fine-tuned model shows three different discrete fossiliferous levels . A firstThe methodology proposed in this study, based on artificially intelligent systems, has been deemed effective in quantitatively identifying discrete fossiliferous levels in paleontological and archaeological sites based on the study of the spatial distribution of fossils. This new, more quantitative approach could be considered a valuable substitute for previous qualitative techniques that have been applied in the past.x axis between the left-hand and right-hand sides of the debris cone, having more weight in the final decision making capabilities of RF than the y dimension. Data of this nature thus supports the need for 3D over 2D analysis in order to ensure an efficient detection of patterns that 2D data may not be able to reveal.Evaluating variable importance through testing for mean decrease in node impurity for RF algorithms  explainsFine-tuned models produced for Batallones-3, in the lower part of a hypothetical hourglass-shaped cavity, clearly show three discrete levels on either side of the debris cone . Cave asThese newly discovered levels in both the lower levels  and upper levels  of the hourglass-shaped cavities of Batallones Butte site support the preliminary inferences made by As previously explained, Batallones-3 deposits and fossils are located in the lower part of the cavity and the fossil site is considered exhausted as the encasing rock was already found, for this reason, the three identified levels can be considered definitive. In the case of Batallones-10, the encasing rock has not yet been found and therefore fossil material recovered in future excavations may reveal additional discrete and separable fossiliferous layers. Thus, the increase in the number of levels at Batallones-10 cannot be discarded.Future studies should analyze the embedded faunal assemblages separately for each of the described levels rather than the entire upper or lower part of the cavity, paying special attention to micromammal remains in order to establish chronological intra-site relationships, such as those inter-site relationships described by The overall workflow of this hybrid intelligence system has proven effective in detecting the presence of 3 levels in both Batallones-10 and Batallones-3. While in some slices the detection of these levels is clearer than in others, the use of two representative, thick and potent slices for each site helps empirically support their identification. Through the implementation of unsupervised algorithms for the initial detection of patterns, expert (geologist)-in-the-loop interactions for sense making and the final tuning of profiles using supervised algorithms, the overall system can be used to find areas where identification of fossiliferous levels is clearer. This can then be extrapolated for the supervised classification of the rest of the site.Moreover, while some isolated points suspiciously appear to be classified into one layer and can be arguably considered out of place, future efforts should try to combine more data for the fine tuning of teaching and training processes. These could include taxonomic or taphonomic variables , data reWhile models and systems without humans-in-the-loop would be an optimal solution for the future , numerouThis study presents a novel use of Artificially Intelligent Systems for the quantitative identification of discrete fossiliferous levels in paleontological and archaeological sites based solely on the study of the spatial three-dimensional distribution of fossil remains. These results have been able to reveal new discoveries in the Batallones Butte site inner structure, including three discrete levels at both Batallones-3 and Batallones-10.The two lowermost levels of Batallones-3, Level I and Level II, dip outwards from the debris cone towards the outer limits of the cave, becoming progressively more horizontal, whereas the uppermost level, Level III is sub-horizontal. On the other hand, Batallones-10’s lowermost Level I dips northward into the cavity and adapts laterally to the cave limits whereas Levels II and III also show northern dips but are more influenced by cave limits and dip towards the center of the cavity in the outermost areas.Through these discoveries, AIAs have been proved to be a highly efficient and objective means of detecting spatial patterns in paleontological sites. Unsupervised and unsupervised techniques can greatly reduce (but not eliminate) the amount of subjectivity in discrete fossiliferous level identification. The possibilities provided by the combination of these algorithms and expert-in-the-loop systems are multiple, however it is important to point out that in order for these methods to be widely used, a need for further experimentation and investigation is essential. This should include an increase in the data used to feed these AIAs. For example, the incorporation of taphonomic features of the remains in the analyzed fossil may provide an even finer tuning of these models.10.7717/peerj.8767/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8767/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.8767/supp-3Supplemental Information 3Click here for additional data file.


Cell migration and invasion is a key driving factor for providing essential cellular functions under physiological conditions or the malignant progression of tumors following downward the metastatic cascade. Although there has been plentiful of molecules identified to support the migration and invasion of cells, the mechanical aspects have not yet been explored in a combined and systematic manner. In addition, the cellular environment has been classically and frequently assumed to be homogeneous for reasons of simplicity. However, motility assays have led to various models for migration covering only some aspects and supporting factors that in some cases also include mechanical factors. Instead of specific models, in this review, a more or less holistic model for cell motility in 3D is envisioned covering all these different aspects with a special emphasis on the mechanical cues from a biophysical perspective. After introducing the mechanical aspects of cell migration and invasion and presenting the heterogeneity of extracellular matrices, the three distinct directions of cell motility focusing on the mechanical aspects are presented. These three different directions are as follows: firstly, the commonly used invasion tests using structural and structure-based mechanical environmental signals; secondly, the mechano-invasion assay, in which cells are studied by mechanical forces to migrate and invade; and thirdly, cell mechanics, including cytoskeletal and nuclear mechanics, to influence cell migration and invasion. Since the interaction between the cell and the microenvironment is bi-directional in these assays, these should be accounted in migration and invasion approaches focusing on the mechanical aspects. Beyond this, there is also the interaction between the cytoskeleton of the cell and its other compartments, such as the cell nucleus. In specific, a three-element approach is presented for addressing the effect of mechanics on cell migration and invasion by including the effect of the mechano-phenotype of the cytoskeleton, nucleus and the cell’s microenvironment into the analysis. In precise terms, the combination of these three research approaches including experimental techniques seems to be promising for revealing bi-directional impacts of mechanical alterations of the cellular microenvironment on cells and internal mechanical fluctuations or changes of cells on the surroundings. Finally, different approaches are discussed and thereby a model for the broad impact of mechanics on cell migration and invasion is evolved. It has been shown that these tugging forces induced and further promoted the invasion of cells into these matrices . These invadopodia can be analyzed in greater depths by either altering the cytoskeletal mechanical characteristics, the mechanical linkage between cellular organelles, such as the nucleus or mitochondria or by probing the surrounding extracellular matrix scaffold directly with mechanical load  or indirectly via the alteration of the structural cues within the matrix scaffold .Generally, to migrate and invade tissues efficiently, cells need to explore and sense their microenvironmental properties, such as matrix properties or fluid flow. Thereby, they need to adapt their own cytoskeletal, nuclear, organelle, or plasma membrane mechanics and alter their surrounding matrix confinement by degradation, secretion of matrix cross-linking molecules, or exert forces to apply tensile stress on their environment. In the following, the matrix environment in Invadosomes can be seen as mechano-adhesive structures involved in the regulation of the invasive capacity of cells through their ability to breakdown the extracellular matrix . The newInvadosomes represent hot-spots of intensified actin polymerization. The invadosome structure is comprised of two continuously polymerizing actin arrangements represented by long pillars of tightly bundled F-actin filaments aligned perpendicular to the substrate. This structure is referred to as the heart of the invadosome structure, in which an actin cluster is formed, consisting of radial F-actin filaments parallel to the substrate . Consequc-Src belongs to the family of tyrosine kinases that possess generally a distinct domain structure, encompassing a myristoylated N-terminal domain that directs Src toward membranes, two Src-homology-protein-binding domains (SH2 and SH3), and a catalytic domain shared by tyrosine-kinases. The activation of Src is facilitated through the impairment of the intramolecular interactions of SH2 with the phosphorylated Tyr527, when specific phosphatases targeting this Tyr residue and thereby causing an “opening” of the entire protein . Apart f–/– mouse embryonic fibroblasts (MEFs), which express an active Src mutant, referred to as SrcY527F, can sufficiently rescue the normal assembly of invadosomes without the capacity to breakdown the extracellular matrix. Apart from the ratio between Tks5/Tks4, the phosphorylation of Tks4 is supposed to be crucial for the governance of this linkage. In fact, the expression of the Tks4Y25/373/508F triple mutant rescues the sustained generation of the invadosome in Tks4–/– MEFs, whereas their breakdown activity is not restored  within invadopodia . The locrestored . These drestored . Thus, irestored .There is much evidence that elevated activity of lamellipodia favored increased 3D migration, invasion and metastasis. In addition, there is ample evidence that lamellipodial regulators, which comprise the compounds of the race activator Tiam-1 and the WAVE complex, are attenuated in metastatic cancer . It is pBone morphogenetic protein (BMP) signaling has been found to stimulate the expression of ARHGEF9b in tip endothelial cells to activate Cdc42 and produce filopodia through formin like 3 FMNL3; . FilopodThere are different types of filopodia, such as long filopodia-like protrusions. It has been seen that a small amount of long filopodia-like protrusions occurs near the periphery of breast cancer cells as they begin to move into the lung parenchyma and interstitium-like environments . Long fiin vivo tissues , encompassing the epidermal growth factor receptor 1, provide the interaction between cell–matrix adhesion receptors and RTKs  and ther tissues ,b. FilopThere exists a number of studies that deal with varying concentrations of extracellular matrix proteins polymerizing into a fibrillar scaffold suitable for the migration and invasion of multiple cell types . When onin vivo  by altering the conditions of the solution, such as pH, temperature, salt concentration, and ionic strength , and secSeveral global transcriptomic or proteomic studies revealed special candidates that foster the onset of colon carcinogenesis due to elevated or reduced expression levels, however, among them are those additionally altered in locally improved or metastatic colorectal cancer . In specin vitro due to sensing of 3D type I collagen , referred to as DDR1 and DDR2 . Each ofin vitro   and MMP-The basement membrane surrounding tissues or vessels and the fibrillar collagen-based interstitial matrix represent physical barriers for cell migration and invasion . A denseEven though leukocytes  apparently migrate protease-independently, mesenchymal cancer cells have to free their route through focusing breakdown activity. MT1-MMP can be considered a membrane-bound matrix metalloprotease that performs a distinct, non-redundant function in the invasion of a number of cancer cell types . In contThe Arp2/3 activator N-WASP has been revealed to fulfill a crucial task in focal proteolytic decomposition of the extracellular matrix. The actin polymerization facilitated by N-WASP encourages the infiltration of MT1-MMP at actin hot spots, which are accumulations of F-actin at places of interaction and contact with the extracellular matrix. MT1-MMP is bound to these actin hotspot herds by means of an actin-binding domain located inside the cytoplasmic tail. Thus, N-WASP-facilitated actin polymerization guides protease activity by producing actin hotspots in immediate vicinity of matrix fibrils intended for breakdown in invasive cells . The WASIn the maturation phase of invadopodia, they accomplish to be proteolytically active, consisting of a process marked by the identification and/or secretion of specific functional MMP enzymes . The thrA hypothesis takes on a necessary function for the membrane-tethered collagenase MT1-MMP through its capacity to proteolytically breakdown matrix components including type I collagen . These rWhen cancer cells expressing MT1-MMP, such as HT1080s cells, are trapped in the dense physiologically networked type I collagen scaffolds, such as 2.0–5.0 g/l type I collagen, and broad-spectrum inhibitors of MMPs are supplemented, no MMP-independent invasion is reported . Cells sin vivo. In contrast, the epithelial cell-specific aiming of MT1-MMP in normal mammary glands leaves the branching unaffected, while the loss of the proteinase in the carcinoma cells reverses the invasion, maintains the matrix structure, and totally prevents metastasis. In contrast, in the normal mammary gland, extracellular matrix reshaping and morphogenesis is only removed when MT1-MMP expression is specifically eliminated from the periductal stroma. These results reveal the complementary but diverse mechanisms that govern the developmental schemes compared to those for redesigning the neoplastic matrix  does not appear to be associated, although it acts as a predominant proteolytic participant in bifurcation morphogenesis and the invasion of carcinoma cells c matrix .Complicated signal transduction cues govern the cancer invasion in 3D extracellular matrices confinements, such as MMP-dependent and MMP-independent mechanisms. Lysophosphatidic-acid-triggered HT1080 cell invasion necessitates MT1-MMP-dependent collagenolysis to obtain matrix voids of nuclear width. These spaces are referred to as single-cell invasion tunnels (SCITs). When SCITs are generated, cells manage to migrate MMP-independent inside them. Apart from cancer cells, endothelial cells, smooth muscle cells and fibroblasts can produce SCITs during their invasion, which leads to the hypothesis that the generation of SCIT is a basic element of cellular motility inside 3D matrices. Precisely regulated signaling actions are necessary for the generation of SCITs. MT1-MMP, Cdc42 and its downstream effectors, including myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) and p21 protein-activated kinase 4 (PAK4), protein kinase Cα and Rho-associated coiled-coil-containing protein kinases (ROCK-1 and ROCK-2) guide the synchronization required for SCIT construction. Finally, MT1-MMP and Cdc42 are fundamental compounds of a cointegrated invasion signaling system that is capable of directing single cell invasion in 3D collagen matrices .in vitro mechano-invasion assay  domains of fibronectin . Since fMultiple cases of integrin crosstalk have been documented in which the attachment of one integrin to its ligand results in a disturbance in the expression or activity of other integrins. This type of crosstalk exists between integrin β3 and another fibronectin-binding family member, integrin β1 . HoweverCofilin activity facilitated actin polymerization is necessary for invadopodia maturation. Thus, knockdown of cofilin is expected to impair the elongation of invadopodia triggered by mechanical stimulation of cells. In line with this, MMP-2 represents a hallmark of invadopodia-based invasion. But it has been found that HT1080 cells have an MMP-independent nuclear piston invasion mechanism that demands integrin β3 activity . It is aper se may increase the invasiveness of another metastatic cancer cell line, the MDA-MB-231 breast cancer cells, or even normal MCF10A epithelial cells (It has been determined that an increased stiffness of the invasion assay matrix (4.5 g/l) had no effect on the increase of invasion through tugging forces compared to a less stiff matrix (2.5 g/l). Besides, an increase in stiffness only, without the tugging force, has not raised the invasion level . Howeveral cells , 2020. HThe p21-activated kinase 1 (PAK1) belongs to the six-member PAK serine-threonine-protein kinase family. It encompasses three major domains: firstly, a kinase domain in the C-terminus, secondly, an autoinhibitory domain and thirdly, a p21-binding domain . The autIn fact, a decrease in integrin β3 signaling due to mechanical stimuli, such as transient tugging force, is connected to the activity of PAK1 . It is wIn human fibrosarcoma cells, the expression of PAK1 is reduced and also its phosphorylation (decreased phospho-Ser144 levels) due to transient stimulation. When integrin β3 is overexpressed, phospho-PAK1 levels are elevated in stimulated cells, indicating that PAK1 is in fact more active. When mutants of PAK1 were expressed in these cells, the so-called kinase-dead mutants displayed enhanced cell invasion, invadopodia maturation and related MMP-2 release. On the contrary, constitutively active PAK1 mutants exhibit a lower invasion, smaller invadopodia and reduced MMP-2 activity. These findings indicate that a reduction in PAK1 activity is required for fibrosarcoma cells to increase their invasiveness as a response to mechanical excitation.Directed cell movement, such as cancer stem cells, provided by contact guidance within aligned collagenous extracellular matrices represents a crucial point for the dissemination of breast cancers throughout tissues . The phyCells can react to mechanical requirements of the local microenvironment by changing their actin cytoskeleton in a dynamic fashion at FAs . In accoThe Ena/VASP family member Ena/VASPlike (EVL) is a new type of regulator of actin polymerization in FAs, and EVL-facilitated actin polymerization has been identified to control cell matrix adhesion and mechanosensors. In addition, EVL plays a decisive part in managing the mechanically directed motility of normal and cancer cells, encourages durotactic invasion and, intriguingly, the inhibition of myosin contractility does not hamper this mechanism. Importantly, suppression of EVL expression interferes with the durotactic 3D invasion of cancer cells. In extension, the reaction to chemotactic  stimulation is amplified in cells with decreased EVL expression, indicating that EVL uniquely facilitates the reaction to mechanical stimuli. A scheme is put forward in which EVL-driven FA actin polymerization strengthens FAs while mechanically paced, which thereby improves mechanosensors, mechanically oriented motility and durotaxis .The expression of MENA or VASP failed to re-establish the diminished adhesion phenotypes in EVL KD cells, and by using chimeric mutants it was determined that the EVH1 domain of EVL is singular amongst the Ena/VASP proteins and is particularly relevant for the proper operation of EVL in FAs. These observational findings reinforce the growing evidence that Ena/VASP proteins, formerly assumed to be exchangeable in functional terms , fulfillDissimilar to intracellular proteins, which are persistently replaced , extrace0, of fibrinogen solutions including or excluding cells during its polymerization has been determined through macroscopic shear rheology. When cells are absent, G0 immediately raises and becomes a plateau after roughly 3 h. This increase reflects the rapid fibrin polymerization to a space-filling elastic scaffolding to be followed by a more retarded process of covalent crosslinking by FXIIIa , it represents a major obstacle for the migration and invasion of cells through narrow subcellular-sized constrictions . The posThe steps comprise firstly the exertion of pressure on the nuclear membrane in the direction of migration due to the external constriction, secondly the incipient deformation of the nucleus due to the formation of a local incident retarding the migration, thirdly the sliding of the compressed and deformed nucleus through the pore and fourthly the backward release, combined with a fast forward pushing and rounding (recoil) of the nucleus . Hence, Subsequently, the nucleus is rate-limiting for the migration of cells through subcellular pores or 3D extracellular matrix scaffolds . The shaThe chromatin packing state changes continuously depending on transcriptional requirements, the level of DNA repair and the specific cell cycle phase. Any of these events bring about modifications in the DNA organization, with temporary and reversible transformations from dense heterochromatin to more open euchromatin due to histone acetylation or demethylation  that conDespite of this, the majority of cells exhibit in 3D substrates rather egg-shaped or spherical shaped nuclei with a diameter between 5 and 15 μm . Under eThe nuclear mechanics have been shown to be regulated by cytoskeletal components, such as actin filaments . Beyond Combined approaches of the three different directions seem to be promising to gain new insights into the mechanical processes and how matrix mechanics, cell mechanics and nuclear and other organelle mechanics are interrelated. There is generally a bidirectional interaction between cells and the surrounding extracellular matrix with a special focus on the mechanical phenotypes of cells and matrices. The ultimate goal seems to be still on tunable mechanical cues applied to 3D engineered matrices with varied biochemical and embedded cell compositions. Another focus of future research will lie on the co-culture approaches, where the surrounding cells, such as cancer associated fibroblasts or macrophages and cancer associated endothelial cells can foster or impair the migration and invasion of cancer cells by possibly affecting mechanical phenotype of cancer cells, including the cytoskeletal as well as the nuclear and other organelle phenotype, or the mechano-phenotype of extracellular matrices.CTM wrote the manuscript and prepared all the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study attempts to understand coronavirus disease 2019 (COVID-19) vaccine demand and hesitancy by assessing the public’s vaccination intention and willingness-to-pay (WTP). Confidence in COVID-19 vaccines produced in China and preference for domestically-made or foreign-made vaccines was also investigated.A nationwide cross-sectional, self-administered online survey was conducted on 1–19 May 2020. The health belief model (HBM) was used as a theoretical framework for understanding COVID-19 vaccination intent and WTP.probably yes intent (54.6%), followed by a definite yes intent (28.7%). The perception that vaccination decreases the chances of getting COVID-19 under the perceived benefit construct  and not being concerned about the efficacy of new COVID-19 vaccines under the perceived barriers construct  were found to have the highest significant odds of a definite intention to take the COVID-19 vaccine. The median (interquartile range [IQR]) of WTP for COVID-19 vaccine was CNY¥200/US$28 (IQR CNY¥100–500/USD$14–72). The highest marginal WTP for the vaccine was influenced by socio-economic factors. The majority were confident (48.7%) and completely confident (46.1%) in domestically-made COVID-19 vaccine. 64.2% reported a preference for a domestically-made over foreign-made COVID-19 vaccine.A total of 3,541 complete responses were received. The majority reported a The findings demonstrate the utility of HBM constructs in understanding COVID-19 vaccination intent and WTP. It is important to improve health promotion and reduce the barriers to COVID-19 vaccination. This study investigated vaccine demand and hesitancy by assessing the intention to vaccinate against COVID-19 and willingness-to-pay. We found that a considerable proportion of the public in China has a definite intention to receive the COVID-19 vaccine; a higher proportion expressed a probable intention. Perceived benefits and barriers to vaccination (namely vaccine efficacy and adverse event concerns) of the health belief model constructs were significant predictors of COVID-19 vaccination intent. A substantial proportion was concerned about fake or faulty COVID-19 vaccines; however, this was not a significant predictor of vaccination intention. The willingness-to-pay for the COVID-19 was determined and was found to be positively associated with income. Higher confidence in domestically-made COVID-19 vaccines was also found in this study. The preference for domestically-made over foreign-made COVID-19 vaccines indicates that a future COVID-19 vaccine developed by domestic companies will receive a favourable response from the public in China.The findings of this study provide useful guidance for tailored interventions to enhance the acceptance of a new COVID-19 vaccine once it is available. Promotional messages framing the benefit of vaccination and concerns about new vaccine safety to enhance vaccine uptake is warranted. The origin of the outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially detected in Wuhan, China in December 2019. The new coronavirus spread rapidly around the world within a month of its onset. On 11 March 2020, the World Health Organization (WHO) declared COVID-19 a worldwide pandemic. By the end of May, the COVID-19 had infected over 5 million people across 215 countries or territories and caused more than 300,000 fatalities worldwide The higher marginal WTP for the vaccine was associated with higher socio-economic factorsMajority expressed confidence in domestically-made COVID-19 vaccineNear two-thirds reported a preference for a domestically-made over foreign-made COVID-19 vaccineThis study examined the applicability of the HBM to investigate the intention to receive the COVID-19 vaccine and WTP. The study was conducted after China had declared that the coronavirus outbreak was largely under control. During the data collection period, May 1–19 2020, the total daily confirmed cases of COVID-19 in China were mainly in the single digits, except on 9 and 10 May, where the total number of cases were 14 and 17, respectively. Of notable importance, our data represent a low level of perceived susceptibility of COVID-19 among the study population, thus warranting public enlightenment in terms of not underestimating the possibility of outbreak resurgence, although the country has successfully flattened the infection curve. Previous research implies that high perceived susceptibility and high-risk perception translate into better preventive actions and are associated with enhanced epidemic control . Thus, sIn this study, despite a large proportion (83.5%) expressing their intention to vaccinate, only about 30% reported a definite intention. A study from Malaysia, a Southeast Asia country, with confirmed COVID-19 cases of only over four thousand and less than 100 deaths during the study period in April 2020, found a higher (94.3%) vaccination intention, of which 48.2% reported a definite intent . A smallA high proportion of a definite intention among participants from the central and southern regions corresponds with high numbers of confirmed cases, indicating that people from regions badly impacted by the coronavirus expressed a higher vaccination intention. Participants in the service occupation expressed higher vaccination intentions, showing their awareness of the need for protection among employees in contact-intensive industries. The fact that China has the world's largest population and being the world’s second-largest economy, a massive surge in demand for the COVID-19 vaccine is anticipated once the vaccine is available. With the intense demand, the COVID-19 vaccination should be prioritized for front-line healthcare workers and vulnerable individuals . MultivaChina has experienced various negative events associated with vaccine malpractices and scandals that have resulted in public lost confidence in vaccines , which pIt is important to highlight that the perceptions of the severity of COVID-19 and the perceived benefits in obtaining the COVID-19 vaccine were also significant predictors of a definite intention for COVID-19 vaccination; however, perception of susceptibility was not a significant predictor. External cues to action were found to be important, namely the provision of comprehensive information about the vaccine when it is available. Hence, imparting adequate information to the public is important, particularly the provision of strong evidence of the safety and efficacy of the vaccine from field trials. This study found that slightly over half reported that they would only take the COVID-19 vaccine if the vaccine is taken by many in the public. Further, participants who noted that they will only take the COVID-19 vaccine if it is taken by many in the public expressed lower vaccination intention. The findings imply that promoting COVID-19 vaccination in the forms of advertorials and testimonials may serve as a cue to action to get vaccinated.This study revealed that most were willing to pay an amount of CNY¥100 [US$14] and CNY¥200 US$28] for COVID-19 vaccine. Although the median WTP of the overall participants was CNY¥200 [US$28], it is important to note that the public from many parts of the country may not be able to afford the vaccine at this price. China’s income inequality ranks among the highest in the world and it is largely due to regional disparities and the urban-rural gap ,33. Furt for COVIAnother positive highlight of this study is the high confidence of the study participants in domestically-made COVID-19 vaccines, and a higher preference for domestically-made than foreign-made COVID-19 vaccines. The new Vaccine Administration Law of the People’s Republic of China, effective December 1 2019, has greatly enhanced regulations in the vaccine industry. The new law stipulates stringent regulatory requirements for researching, producing, distributing, and using vaccines. The new law also imposes tough penalties for violating regulations on the production and sales of vaccines . The impEfforts are needed to promote high definite intention to get vaccinated against COVID-19 when the vaccine is successfully developed and given regulatory approvalPublic health intervention programmes should focus on increasing the perception of the benefits of COVID-19 vaccination and reducing the perceived adverse effect and inefficacy barriersClinical evidence of the safety and efficacy of COVID-19 vaccines are key messages to enhance rates of vaccine coveragePromoting COVID-19 vaccination in the forms of advertorials and testimonials may prompt vaccination decisionIt is crucial to reduce inequalities in access to COVID-19 vaccines due to financial constraintsFurther measures are needed to identify and restore confidence in domestically-made COVID-19 vaccines among certain individuals and societal groupsThere are some limitations of the current study that need to be considered in interpreting the results. Firstly, the use of an online survey may result in sampling bias, so results may not be generalizable to the wider community as reflected in lack of representative from some provinces. The intrinsic disadvantages of Web-based surveys concern the generality and validity of results warranting careful interpretation of the research findings ,37. It iHealth promotion is warranted when the COVID-19 vaccine is available because, among those who expressed intention to be vaccinated, over half expressed probable intention. The HBM model can be used to develop strategies for enhancing COVID-19 vaccine uptake and WTP. Most importantly, the findings revealed that interventions targeting perceived barriers, i.e. the efficacy and adverse events related to a new COVID-19 vaccine, are most crucial. Concerns regarding faulty or fake vaccines were not a significant predictor for COVID-19 vaccination intent, perhaps implying public trust in the government’s new vaccine administration law.The WTP was positively related to socio-economic factors, which should provide guidance for policy recommendations for the future national COVID-19 improvisation program in mainland China.S1 Text(DOCX)Click here for additional data file.


When imaging brain vasculature with optical coherence tomography angiography (OCTA), volumetric analysis of cortical vascular networks in OCTA datasets is frequently challenging due to the presence of artifacts, which appear as multiple-scattering tails beneath superficial large vessels in OCTA images. These tails shadow underlying small vessels, making the assessment of vascular morphology in the deep cortex difficult. In this work, we introduce an image processing technique based on mean subtraction of the depth profile that can effectively reduce these tails to better reveal small hidden vessels compared to the current tail removal approach. With the improved vascular image quality, we demonstrate that this simple method can provide better visualization of three-dimensional vascular network topology for quantitative cerebrovascular studies. Optical coherence tomography (OCT) is a high-resolution imaging modality used to visualize the microstructure of living tissues, such as the human retina , and wast, however, the light passing through the vessel also undergoes dispersion but the shape of the wavefront distortion is altered because the position and density of RBCs change as they continue to move. Consequently, the optical path of light backscattered from the tissue changes after Δt, resulting in temporal decorrelation in the OCT signal (tissue(t)E) at the same tissue location. This amplitude or phase difference as OCT signals travel through static tissue results in residues that appear as tails extending downward from the vasculature. Behind pial vessels with high hematocrit levels, in particular, these spurious projections can extend to the lower depths of the cortex, overshadowing true vessels. This shadowing by superficial large vessels remains as ghost images in deeper regions, complicating the delineation of vascular networks for different cortical layers.Although OCTA offers three-dimensional (3-D) vascular information, morphologic analysis of vessels is often limited to two-dimensional (2-D) projections of the volumetric vasculature because of tail artifacts. These imaging artifacts are observed as dynamic multiple scattering that appears beneath the blood vessels in cross-sectional OCTA images . Figure In order to reduce tail artifacts, a few engineering and software methods have been reported. Leahy et al. employed OCT with a high numerical aperture objective to achieve a shallow depth of field that is much smaller than the scattering length, which essentially blocks multiple scattering, thus mitigating tail artifacts . Despiteen face OCT structural image with OCT angiograms. However, the multiplication of OCT slab might induce signal loss of capillaries not underneath the large vessels because of speckle textures seen on the en face OCT image [Furthermore, several image processing methods, including step-down exponential filtering ,20 and sCT image .In this paper, we introduce a practical approach based on mean subtraction for the removal of tail artifacts from cerebral OCTA images. Compared to existing methods, our thresholding method is adaptive based on discrete local tails, thus mitigating individual tails and balancing signal loss for capillaries behind very large vessels against the preservation of low intensity information in deeper areas. For brain imaging in mice, the proposed method preserves information about blood flow in the deep cerebral cortex, providing better angiographic contrast than existing methods.Animal protocols were approved by the animal care committee at the University of Washington (protocol number: 4262-01). After being placed under anesthesia using 1.5–2% isoflurane , three female C57/BL6 mice weighing 23–25 g  and immobilized in a stereotaxic frame were surgically implanted with cranial windows. A standard cranial window ~3 mm in diameter was centered ~1.5 mm posterior to the bregma and ~1.5 mm lateral to the midline. An open-skull cranial window was used to increase the optical penetration depth of the tissue, as described previously . To creain vivo imaging of the mouse cerebral cortex. The light source  consisted of two super-luminescent diodes combined by using a 10/90 fiber coupler to obtain a bandwidth of 110 nm, providing an axial (depth) resolution of 7 μm in air (~5 μm in brain tissue with a refractive index of ~1.35). A 1340 nm spectral/Fourier domain OCT microscope, shown in The use of a 10× objective in the sample arm resulted in a transverse intensity profile with a full width at half maximum value of 7 μm at focus, corresponding to lateral resolution. The output light from the interferometer was routed to a home-built spectrometer with a 2048 pixel InGaAs line scan camera  operating at 92,000 axial scans/s. The system had a measured sensitivity of 105 dB with an optical power of 3.5 mW on the sample surface.A line trigger from the camera was used to synchronize the output of the galvanometer drive signals, ensuring that axial scans in repeated frames were acquired at the same transverse location. The operations for beam scan, data acquisition, data storage, and hand-shaking between the procedures were fully controlled using a custom software program written in the LabView language.2en face image (angiogram) projecting all vessel information at specific depth ranges was finally obtained.In order to visualize microvasculature structures through the cranial window, we used optical microangiography (OMAG), an amplitude-based OCTA method . For theDS(i)OCTA and OCTA(i) are the i-th A-lines of the de-shadowed image and the original image, respectively, and OCTA is a magnitude at a k-th pixel point of the i-th A-line of the original image. N = 1024 is the total number of pixel points of the A-line, and w is a constant weighting the mean value. Unlike a purely thresholding-based method [We propose the use of a simple mean-subtraction method to remove the tail artifacts in OCTA mouse cortex images. The mean-subtraction algorithm isd method , this alOCTA is an OCTA signal at an arbitrary depth position z of the i-th A-line of the original image and γ is a proportionality constant that controls the rate of attenuation. DSOCTA of the exponent is a k-th pixel point of the i-th A-line, some of which were de-shadowed from the top surface to z−1. For comparison with the mean-substitution method, the two proportional constants w, γ in Equations (1) and (2) were manipulated to have nearly equal attenuation of tail artifacts, and the de-shadowed images were normalized without any contrast adjustment for direct comparison. The step-down exponential filtering method ,20, whicTo understand the progression of cerebral vascular disease with OCTA imaging of the cerebral microvasculature, it is important to capture the true vessel density distribution as the vessel density varies throughout cortical layers. Here, we used single cross-sectional OCTA images of the mouse cerebral cortex obtained through a cranial window as described in the Methods section to evaluate our algorithm. The original image a shows sw of 1.5, 2.0, and 2.5 is shown in w of 2.0. Graphing a section of a single capillary  processed with the step-down method. To compare the effect of the processing methods on image contrast, the root-mean-square (RMS) contrasts of 200 horizontal line intensity profiles taken from each angiogram (440–460 μm) of The proposed algorithm is a simple and effective approach to remove tail artifacts in deeper layers of OCTA images of the mouse cortex. However, a limitation of this approach is that it may induce partial loss of capillaries close to the middle of the tail artifact in OCTA images. At points closer to the center of the vessel lumen, the tail below the vessel becomes longer, increasing the mean value of the line. The subtraction of the mean value, therefore, may cause the loss of capillaries with signals below the mean value. This effect is more significant for capillaries below larger surface vessels (>100 μm) or for deeper capillaries with weaker signal strengths. This limitation might be mitigated with the help of engineering techniques that can physically reduce the tail artifact; for instance, an alternative hardware-based method, dynamically focused optical coherence microscopy angiography with a high numerical aperture, has successfully been applied to reduce the tail artifacts in synthesized angiograms . Anotherin vivo. The proposed algorithm noticeably increases the contrast in cross-sectional images (en face images (The brain contains a complicated vascular network that varies in density throughout the cortical layers . Within l images b as welle images b, resultIn conclusion, we introduced a simple post-processing method based on mean subtraction to effectively suppress vessel tail artifacts in microvascular angiograms, preserving contrast in deeper vascular regions. Application of this method to volumetric OCT angiography images of the mouse cerebral cortex obtained through a cranial window led to the reduction of tail artifacts and enhanced the contrast for parenchymal capillary vasculature. In the future, this method may be further applied to quantitatively measure changes in vessel density or tone, which may have applications beyond the neuroimaging field, such as in ophthalmology.


The SARS-CoV-2 infection was declared as a pandemic by the World Health Organization (WHO) on March 11, 2020, and the death toll from COVID-19, which is the disease caused by SARS-CoV-2, has already surpassed that of many previous epidemics. A wide variety of treatment options are being considered for COVID-19, but there is still no definitive treatment or vaccine. This study aims to explain the background of convalescent plasma (CP) treatment and its relations with COVID-19 immunity, to define ideal treatment procedures, and to reveal present and future perspectives in the light of the rapidly growing data.Since it has been shown that the impaired immune response of the host is one of the most important factors that increase the severity of the infection, treatment strategies to suppress aberrant immune activation are currently being considered. CP, which is derived from recently recovered patients and contains neutralizing antibodies and many other immunemodulatory substances, seems to be the most convenient strategy to restore normal immune function considering the fast spreading nature of the ongoing pandemic.Even though mechanisms of action of plasma therapy are not fully delineated, it was shown that it could lead to a reduction in mortality since other alternatives such as monoclonal antibodies or SARS-CoV-2 hyperimmunoglobulin require much more time and effort to be developed. Visualizing the History of Pandemics [online]. Website u1d8b [accessed 20 June 2020].. On March 11th of 2020, the World Health Organization (WHO) declared that the viral infection and subsequent outbreak ofSARS-CoV-2 constituted a pandemic2World Health Organization (2020). WHO announces COVID-19 outbreak a pandemic [online]. Website u1d90 [accessed 20 June 2020].. ‘Pandemic’ is the term used for epidemicscharacterized by multiple, infectious casesin distinct countries and continents. COVID-19 and the associated immune syndrome is a multisystemic disorder following the development of the SARS-CoV-2 infection. Currently, the death toll of COVID-19 has already surpassed those of previous epidemics such as SARS-CoV-1, MERS, Ebola, Yellow Fever, and the H1N1 influenza virus and mortality numbers are still growing1.The term ‘epidemic’ comes from the conjugation of the Greek words ‘epi’ (on) and ‘demos’ (people) and was first mentioned by Homer. Hippocrates first used this term in the field of medicine to describe an assortment of clinical syndromes occurring and spreading at a certain location in a given period [1]. Over time, the term has been utilized to describe outbreaks of single infectious diseases, such as the Bubonic Plague (Black Death), which caused the deaths of approximately 200 million people between 1347 and 1351A wide variety of treatment options are currently being considered for the challenging COVID-19-associated immune syndrome. From an immune-biological point of view, the impaired/deficient immune reaction of the host could play a prominent role, particularly in patients presenting with more severe clinical diseases. Drugs and treatment options focusing on immune modulation of the host are currently under development. Convalescent/immune plasma (CP) derived from recently recovered patients is a classical way of passive immunotherapy experienced within the last 80–100 years [2]. Since the SARS-CoV-2 specific hyperimmune immunoglobulin preparation or unique monoclonal antibodies require much more time and effort to be developed, CP, which contains neutralizing antibodies to the relevant pathogen [3], is the most convenient way, considering the fast spreading nature of the ongoing COVID-19 pandemic until an effective vaccine becomes available.Currently, available data indicates that about 80% of SARS-CoV-2 infections are asymptomatic or mildly symptomatic, whereas the remaining 20% can manifest itself as the severe disease form. The host immune system, together with the pathogen virus dose, viral strain, time-dependent viral transmission, and host exposure kinetics determine the severity of the disease. Since it is a newly emerging disease, precise data regarding the immunological aspects of the COVID-19 syndrome are still being collected. The most analogical explanations are being made based on previous members of the coronavirus family viruses, namely SARS-CoV and Milddle East respiratory syndrome (MERS).[4].+, CD8+ T-cells, and antibody-producing B-cells is involved, and a second wave of inflammation takes place [4]. Coronaviruses could also further lead to lymphocyte apoptosis, another well-known strategy for immune evasion [6].Innate immune system and type-1 interferon molecular expressions, along with other proinflammatory cytokines, including IL-1, IL-6, and TNF-alpha, play a central role in the clearance of coronaviruses [5]. The severe disease forms of SARS-CoV, MERS, and SARS-CoV-2 are associated with immune evasion strategies. These biological events enable the viruses to proliferate in the infected host cells. Eventually, the infected cells undergo programmed cellular death. Meanwhile, the viral particles, along with the intracellular components, such as nuclear antigens triggering the innate inflammatory mechanisms, are released into the microenvironment. At this critical biological stage, adaptive immunity with CD4In a subpopulation of infected patients with SARS-CoV-2, enhanced immune activation could lead to a cytokine storm, which clearly contributes to disease severity with increased morbidity and even mortality [7]. The reduction in the total number of T-cells was demonstrated in patients with severe COVID-19-associated immune syndrome requiring intensive critical care. T-cell numbers are negatively correlated with the circulating IL-6, IL-10, and TNF-alpha. Patients with a history of recovering fromthe COVID-19 syndromeshowed a reduced concentration in proinflammatory cytokine levels together with increments in the circulating T-lymphocyte counts. Moreover, significant increases in exhaustion markers such as PD-1 were observed on the T-cells, which became more apparent during the clinicobiological course of the COVID-19 associated immune syndrome [8]. Many systems in the body are affected by the direct cytopathic effect of the virus or one/several of these immune mechanisms. Some of the clinical and pathological manifestations are demonstrated in Figure 1. Although there is much to still learn about the immune pathology of this newly emerging syndrome, the impaired and exaggerated immune response seems to be the most important mechanism that increases clinical severity. Therefore, treatment strategies to suppress aberrant immune activation and restore the normal function of the immune system are urgently needed.Passive immunotherapy dates back to the late 19th century, when patients with diphtheria were treated with the immune sera of animals such as sheep, goats, or horses. However, the use of human CP has been in practice since the early 20th century with the emergence of viral outbreaks such as the 1918 H1N1 influenza virus pandemic [9]. The immune plasma transfer approach was also used successfully during the 2003 SARS-CoV epidemic [10], the 2009–2010 H1N1 influenza virus pandemic [11], the 2012 MERS epidemic [12], and the 2014 West African Ebola epidemic [13]. In a systematic review and exploratory metaanalysis by Mair-Jenkins et al., the administration ofCP for the treatment of severe acute respiratory infections of viral etiology was assessed. The authors concluded that, in spite of the lack of randomized controlled clinical trials, plasma treatment could lead to a reduction in mortality, especially when it is administered in early periods following the onset of the infection [14]. This observation can be partly explained by the smaller amount of initial inoculum. Since the viral load in the initial inoculum is much lower than that of the established infection, it makes it easier for the passive antibodies to neutralize them. Viral neutralization and relevant cellular immunological mechanisms can operate more efficiently at the beginning of the immune response [15].Several mechanisms are now being proposed for the mechanism of action ofCP therapy. Neutralizing antibodies, which bind to the virus and block viral entry, fusion, and egress, are the most recognized mechanism [16]. A dose of neutralizing antibodies is correlated with higher viral load reduction and lower mortality, as suggested in previous studies on the Ebola virus and the Junin virus, the causative agent of Argentine hemorrhagic fever . The fact that it contains antibodies that neutralize the virus and block its entrance to host cells by binding to crucial viral antigens is not the only mechanism of action ofCP. Immune plasma therapy may also function via modifying the host inflammatory response and related endogenous substances. + T cells, and Th1- and Th2-associated cytokines from infection . Likewise, FrCasE retrovirus-infected mice to which the passive antibody was administered had protective immunity in spite of a lower viral inoculum mimicking the neutralizing function of the antibody [23]. Those in vivo observations generated the hypothesis that passive antibody administration could lead to similar immunobiological effects in humans. That hypothesis will be tested while performing future research during the ongoing COVID-19 pandemic because, at present, there is no available and effective vaccine or antiviral drug.The efficacy of CP is dependent upon distinct immunobiological actions [19]. Numerous cellular responses are involved in the immunological responses, including T-cells, B-cells, dendritic cells, and macrophages. They are all involved in the ‘cellular immunity effects’ of CP via modulation of cytokines and chemokines within the maturation and activation of each unique cell type. Although these mediators and their clinical effects have not yet been exactly ascribed to direct CP contents and in vivo effects, many postulations have been made regarding the already known mechanisms of IVIg treatment, which acts through similar pathways. Antibody-dependent cellular cytotoxicity, opsonization, and complement activation take part in humoral immunity. These complicated biological processes could aid antibodies in stimulating cellular immunity within the actions of cytokine/chemokine networks [20]. Passive antibody administration failed to protect mice deficient in CD4Another important problem in the course of COVID-19 is coagulopathy, which often occurs in findings similar to disseminated intravascular coagulopathy or antiphospholipid antibody syndrome. Critically ill patients exhibited positivity for anticardiolipin IgA antibodies, aswell as for anti-β2-glycoprotein I IgA and IgG antibodies [24].CP therapy may also have a role in terms of blocking responsible antibody for prevention or amelioration of antiphospholipid antibody syndrome-like coagulopathy, just as this has been observed in IVIg therapy.3U.S. Department of Health and Human Services Food and Drug Administration (2021). Investigational COVID-19 Convalescent Plasma [online]. Website u1d92 [accessed 00 Month Year]..The efficacy of CP therapy has not yet been proven by randomized controlled clinical trials. According to preliminary data, the most beneficial way to use immune plasma therapy is within the first 10 days of the disease course and in COVID-19 patients following severe or life-threatening disease criteriaSevere disease criteria:- Dyspnea- Respiratory rate ≥30/min- Arterial oxygen saturation ≤93%2 ≤ 300- Pa/FiO- >50% progression of lung infiltrates within 24–48 hLife-threatening disease criteria:- Respiratory failure- Sepsis or septic shock- Multiorgan dysfunction or failureInformed consent must be obtained from the patient or from the patient’s relatives.4World Health Organization (2021). WHO Blood Regulators Network (BRN) Donor Selection in case of Pandemic Situations [online]. Website https://www.fda.gov/media/136798/download [accessed 20 June 2020]..Individuals who donate immune plasma must first be in good health; meeting universal blood donation criteria and adhering to standard operating procedures prior to the pandemic are also important. As with many other blood product donations, screening should be done for the main viral infection factors that can be transmitted by blood. The main essentials of those tests include HIV 1-2 and HIV NAT, anti-HCV-HCV NAT, HBs Ag, and VDRL but should also include regional infectious diseases, if any. Special warnings regarding blood donation during the pandemic period have been specifically stated by WHO5European Commission Directorate-General for Health and Food Safety (2020). An EU Programme of COVID-19 Convalescent Plasma Collection and Transfusion [online]. Website https://www.who.int/bloodproducts/brn/DonorSelectionincaseofPandemicSituations.pdf?ua=1 [accessed 20 June 2020].:The following criteria regarding COVID-19 disease must be met- During the disease period, a diagnostic test must be positive  or a SARS-CoV-2 serological antibody test must be found to be positive after recovery.- Complete COVID-19 symptoms have to be resolved at least 14 days before donation (there is no need to prove that the PCR test has become negative)- Donation must be from male donors or nonpregnant female donors or anti-HLA antibody negativity in women with a history of pregnancy- Testing of anti-SARS-CoV-2 antibody titers, if available:- Ideally, antibody positivity should be found at a titer of 1:160 or higher according to the FDA and 1:320 or higher according to European Commission on Health and Food Safety. If no alternative is available, a lower titer may also be effective.- If the test is not available, it is recommended that the plasma sample be stored for testing later when available.It is essential that all these processes be carried out in authorized and high standard blood banks. The schematic steps of CP treatment are demonstrated in Figure 2.Although it is not yet clear whether the SARS-CoV-2 virus is transmitted by blood and since plasma products should not contain any nucleic acid-containing cells (leukocytes or microorganisms), inactivation of possible pathogens  should be performed in each donated plasma product in centers with the technical abilities and trained personnel. For this, solvent/detergent-based treatments that are very effective against viruses with lipid envelopes or light-based methods targeting the nucleic acid structure  can be used. Pathogen-reduced plasma products have not been observed to cause any toxic or immunologic adverse reactions beyond what would be expected from any other plasma products.It is recommended to label the donor’s ABO and Rh groups, the date of donation, the amount of plasma contained, and the antibody test result for the patient it is prepared for [25].A 55-year-old male with a history of myelodysplatic syndrome (MDS) with fluorescent antibody test (FAB) refractory anemia excess blasts-1 subtype (MDS/ RAEB-1), complicated by disseminated systemic tuberculosis and associated kidney disease, was admitted to our hospital with complaints about an ongoing, high fever and persistent cough lasting for about 3 days. He had recently been discharged from the hospital after a follow-up of 2 months for the disseminated tuberculosis infection and was still on the classic four-drug regimen . Low-dose chest computed tomography indicated COVID-19 pneumonia , and the nasopharyngeal swab sample was also found to be positive for the COVID-19 infection; he was then urgently hospitalized. Taking into consideration the immunocompromised status of the multimorbid patient and his worsening status, despite antiviral (favipiravir) treatment and supportive care, 200 mL of CP was transfused twice at 2-day intervals. In addition, to break the severe macrophage activation syndrome-like immune response, tocilizumab was administered with the 2nd infusion. In the following period, the patient rapidly improved in terms of clinical and laboratory values. Timely infusion of CP in this patient, who had difficult comorbidities and immune compromisation and an uncontrolled immune response similar to macrophage activation syndrome, is a demonstrative example [26].6U.S. National Library of Medicine (2020). ClinicalTrials.gov Database [online]. Website https://clinicaltrials.gov [accessed 00 Month Year].. In those studies comparing various comparators with immune plasma, many parameters, especially effectiveness and safety, were evaluated in terms of a wide range of clinical outcomes. Studies involving more than 100 patients are summarized in the Table. The results of those studies show the use of immune plasma and with which indications and timing it will be most beneficial. However, some of the most important publications on immune plasma effectiveness [27] and safety [28] are already at the preprint stage. The safety data in one study, which involved 5000 patients, indicated that immune plasma administration could be considered safe in those with severe or life-threatening diseases. In another study, in which both efficacy and safety were addressed, immune plasma treatment was evaluated as safe, and clinical improvement was achieved in 19 of 25 patients [27]. No significant adverse effect was observed in a study in which 26 patients were included in a center where the CP treatment was first applied in a country outside of China. Six of 17 patients who needed mechanical ventilation died, while none of the 9 patients who did not require mechanical ventilation died [29]. As more data from more studies accumulate, more robust conclusions will be drawn regarding immune plasma efficacy and safety.In many countries, large-scale CP studies have started to be carried out, some of which have involved more than 1500 patients7U.S. Food and Drug Administration (2020). Coronavirus (COVID-19) Update: FDA Coordinates National Effort to Develop Blood-Related Therapies for COVID-19 [online]. Website https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-coordinates-nationaleffort-develop-blood-related-therapies-covid-19 [accessed 20 June 2020].. Plans are in motion to purify polyclonal antibodies such as hyperimmune globulin (H-Ig) from pooled plasma of donors to develop an unbranded standard antibody product. With the production of hyperimmune globulins, an antibody-based treatment will not have to be stored locally but can be shipped to many locations worldwide. These types of treatment will also offer a standardized and highly efficient, scalable option, and clinical outcomes can be tracked more objectively. If the expected success is achieved in the previous experiences, then steps towards the production of recombinant antibodies in mammalian cell lines can be envisaged.In the near future, antibody-based treatments will not only constitute CP


Immune checkpoint inhibitors (ICI) have revolutionized the treatment of cancer worldwide. Not long ago, before the introduction of ICIs, many cancers incurred a grave prognosis on patients due to the lack of effective therapies. For instance, patients with malignant melanoma survive longer and experience a better quality of life than ever before due to agents such as nivolumab and ipilimumab. Nevertheless, toxicities associated with the use of ICIs have been increasingly recognized in clinical trials as well as oncology practice. The widespread usage of ICIs and the expected addition of newer ICIs to the arsenal of medications to fight cancer raise awareness of the potential toxicities of these medications. Once these toxicities develop, immunosuppression with or without withholding immunotherapy is the standard of care. Because the long-term adverse effects of these toxicities and the impact of stopping therapy on survival are not well characterized, a joint decision by both the oncologist and the patient should be carried out if stopping therapy is being considered. Nevertheless, long-term data is necessary to guide such decisions. In this article, we will discuss common ICI’s immune-related adverse events with a simplified approach to recognizing and managing these events.  Immune checkpoint inhibitors (ICI) are monoclonal antibodies that activate the immune system by inhibiting agents that downregulate the function of T-cells. ICIs block several targets, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and programmed death ligand1 (PD-L1), which in turn disinhibit proliferation of antitumor T-cells . While bThe development of “immune-related adverse events” (irAEs) is thought to be secondary to the enhancement of the immune system activity against tumor cells. The resultant autoimmunity and inflammatory reaction against host-tissue mediated by T-cell, antibodies, and cytokines largely responsible for such toxicities . ExacerbThe significance of irAEs stems from the fact that up to half of the patients who receive ICIs experience these side effects . In factWhile irAEs affect any organ system, dermatologic, gastrointestinal, pulmonary, endocrinological, hematologic, and musculoskeletal adverse events are among the most commonly reported . The irAThe Common Terminology Criteria for Adverse Events (CTCAE version 5) was designed initially to classify toxicities from chemotherapy, but it is largely used to grade toxicities resulting from immunotherapy as well . The criGastrointestinal immune-related adverse effectsColitis is one of the most common irAEs, and the incidence of colitis ranges between 5% for PD-1 and PD-L1 and 25% for CTLA-4 . Before Hepatitis is suspected in the elevation of liver enzymes and/or bilirubin levels. Elevation of liver enzymes occurs in 1% of patients on anti-PD-1/PD-L1 and in 10 % on anti-CTLA-4 agents . PatientPulmonary immune-related adverse effectsAffected patients usually present with pneumonitis. Patients with pneumonitis attributed to ICIs typically have evidence of diffuse inflammation of the lung parenchyma on computerized tomography (CT) imaging of the chest ,2,8. ExcRheumatic immune-related adverse effectsThe largest single-center cohort of rheumatic immune-related adverse effects (Rh-irAEs) studied to date is a retrospective analysis done on a cohort of 1293 patients who received ICIs. Sixty-one patients developed Rh-irAEs with a prevalence of 4.7%, and 14 patients had preexisting documented autoimmune diseases . While tEndocrine immune-related adverse effectsRetrospective analysis data done on patients who received ipilimumab in a single comprehensive cancer center found that the most commonly encountered endocrinological irAEs were hypophysitis and thyroiditis, with an overall incidence of 8% and 6% respectively . InvestiNeurologic immune-related adverse effectsNeurological irAEs are rare with an incidence of <1% of patients treated with ICIs . HoweverDermatologic immune-related adverse effectsSkin toxicities occur in up to 30% of patients treated with ICIs and may range in clinical presentation from pruritus and acneiform rash to toxic epidermal necrolysis ,8. BlistDue to the lack of prospective clinical trials to guide the management of immune-related adverse effects of immunotherapy, expert opinions based on observational studies have driven the development of standardized guidelines for the management of these side effects. There are available clinical practice guidelines to help clinicians and patients decide on the management plan, and a multidisciplinary team approach is recommended. In general, grade mild toxicities can be managed by the temporal cessation of the offending medication and therapy can be restarted once toxicities resolve. Moderate and severe toxicities may require the administration of systemic steroid treatment and/or another immunosuppressive regimen in addition to the permanent cessation of immunotherapy for the majority of severe toxicities. Endocrinological adverse reactions can be managed with hormone replacement, and immunotherapy can be continued. Other immunosuppressant agents, such as infliximab and mycophenolate mofetil, are beneficial in patients refractory to steroids.


The development and deployment of a web-based, self-triage tool for severe respiratory syndrome coronavirus 2 (COVID-19 disease) aimed at preventing surges in healthcare utilization could provide easily understandable health guidance with the goal of mitigating unnecessary emergency department (ED) and healthcare visits. We describe the iterative development and usability testing of such a tool. We hypothesized that adult users could understand and recall the recommendations provided by a COVID-19 web-based, self-triage tool.We convened a multidisciplinary panel of medical experts at two academic medical schools in an iterative redesign process of a previously validated web-based, epidemic screening tool for the current COVID-19 pandemic. We then conducted a cross-sectional usability study over a 24-hour period among faculty, staff, and students at the two participating universities. Participants were randomly assigned a pre-written health script to enter into the self-triage website for testing. The primary outcome was immediate recall of website recommendations. Secondary outcomes included usability measures. We stratified outcomes by demographic characteristics.A final sample of 877 participants  was used in the analysis. We found that 79.4% of the participants accurately recalled the recommendations provided by the website. Almost all participants (96.9%) found the website easy to use and navigate.Adult users of a COVID-19 self-triage website, recruited from an academic setting, were able to successfully recall self-care instructions from the website and found it user-friendly. This website appears to be a feasible way to provide evidence-based health guidance to adult patients during a pandemic. Website guidance could be used to reduce unnecessary ED and healthcare visits. The severe respiratory syndrome coronavirus 2 COVID-19 disease) pandemic has led to more than 287,399 deaths worldwide.9 diseaseA key strategy to mitigate the healthcare system burden is “forward triage.”5flu.gov website as well as Microsoft Corporation’s H1N1 Response Center.www.c19check.com). The original SORT algorithm has been updated to reflect the best available evidence about COVID-19 as shared by the Centers for Disease Control and Prevention and the World Health Organization, and was built iteratively with multidisciplinary input from experts in infectious disease, emergency medicine, pre-hospital medicine, epidemiology, and health literacy. On the website the user enters age, ZIP code, comorbidities, and symptoms, and the algorithm classifies risk as low, intermediate, or high (Web-based self-triage was first developed and deployed during the 2009 novel influenza A (H1N1) virus pandemic by researchers at Emory University. The evidence-based Strategy for Offsite Rapid Triage (SORT) was rapidly validated and integrated into web platforms hosted on the US Department of Health & Human Services  or high . The webThe primary objective of this study was to determine whehter participants understood and recalled recommendations provided by a COVID-19 web-based, self-triage tool.Investigators at Emory University and Uniformed Services University of the Health Sciences performed usability testing of the COVID-19 self-triage website. The institutional review boards approved this study and waived the consent requirement; however, an assent script with affirmation was used. On March 19–20, 2020, staff, faculty, and students at both institutions were recruited via email for voluntary study participation. Participants received an email with instructions and a link to the self-triage website. After completing demographic information, each participant was randomly assigned a pre-written patient “script” that the participant referred to when using the COVID-19 checker website. Once participants inputted scripted information into the COVID-19 checker website and received a recommendation, they were directed to complete the user-feedback form. This form contained questions about their understanding of the recommendations, and their subjective experiences using the website.We excluded participants’ responses if they did not complete all fields in the website or the user- feedback form. The website is currently designed to triage adult patients. To test the software, some participants were randomly assigned clinical scripts with patients younger than 18 years old. These cases were not allowed to progress to the clinical questions or website guidance and were therefore removed prior to endpoint analysis.The study occurred virtually with participants at Emory University and Uniformed Services University of the Health Sciences.Staff, students, and faculty at Emory University and Uniformed Services University of the Health Sciences participated in the study.We assessed participants’ ability to immediately recall the specific self-care instructions provided by the site, scored as either correct or incorrect. Self-care instructions were specific and nuanced, depending on the symptoms and comorbidities of the scripted case provided. Secondary outcomes were participant feedback about website usability.A total of 926 participants enrolled in the study. One participant was removed for being younger than 18 years old, and participants who received pediatric scripts (n = 46) or missing scripts information (n = 2) were removed from analysis, leaving 877 participants  in the final analyses . We founThis study demonstrates the feasibility of a web-based, self-triage tool to provide information to adults seeking guidance about COVID-19 symptoms and next steps. Approximately four out of five participants correctly recalled and identified the website’s recommendations, and nearly all participants described the website as easy to use.The sampling methodology allowed for rapid feedback of the website, and free-text suggestions were expeditiously incorporated into the website. Suggested modifications were incorporated the day after study enrollment, and the website was launched for public use the following day. Using the prior H1N1 website experience, the current COVID-19 algorithm was created, rapidly iterated, adapted, tested, and electronically deployed within days of sustained community transmission in the US, and well before the anticipated zenith of COVID-19 in the US.C19check.com has hosted 766,574 unique visitors and completed 395,895 self-assessments. In comparison, there were 320,333 unique visits to the H1N1 self-triage website during its first three months. The self-triage algorithm will be updated in real time as new guidance and data are published by national and international public health experts, and in continued consultation with health literacy experts. Future studies will validate the algorithms and test user understanding of self-assessment instructions, adherence to instructions, and intent-to-use healthcare resources before and after self-triage within the wider population of website users. Website data will also be used to map epidemiologic patterns of disease and symptoms and will continue to expand upon the list of 15 foreign-language translations available.Thus far, there is high public demand for this self-triage tool. Between March 26–May 14, 2020, The participants were students and faculty at two academic institutions and thus the results cannot be applied to the general population due to mismatched health literacy and recall capacity. Given the exigencies of the COVID-19 pandemic, these sites were chosen since a large number of participants could be recruited rapidly, and the free-text feedback from medically-oriented participants was instructive for the design team. For this reason, we chose to capture a mix of free-text usability feedback and Likert-scale usability scores rather than a validated usability scale for the secondary outcome. This study tested immediate recall of instructions, rather than lasting recall of instructions, as the website was designed to be used by a person actively making the decision to seek medical care or not, and the instructions would be less relevant at a later date.Additionally, the self-care instructions were nuanced and specific by design, which could have negatively impacted recall success depending on the comorbidities and symptoms in the user’s assigned scripted case. Further, the scripted nature of the cases could also have had a negative impact on recall success. This study did not seek to identify adherence to the instructions  since the participants were not actually using their own real patient data. The study did not assess the outcomes of actual COVID-19 patients following recommendations from the site.This study demonstrates that adult users in an academic setting can correctly identify recommended care instructions from a self-triage website during a pandemic. Study participants found the website user-friendly. The website was adapted from a pre-existing, self-triage algorithm in an iterative, expeditious manner. To date, there has been high demand for the website, and it has potential to provide users valuable health information and mitigate unnecessary ED visits. Limiting unnecessary healthcare visits will benefit both patients and healthcare workers by reducing COVID-19 exposure while conserving scarce resources.