Petromyzon marinus and Lethenteron camtschaticum) and two genes in a hagfish (Eptatretus burgeri). Synteny analyses show that all five lamprey CRH-family genes have similar chromosomal neighbors as the gnathostome genes. The most parsimonious explanation is that the lamprey CRH-family genes are orthologs of the five gnathostome genes and thus arose in the same chromosome duplications. This suggests that lampreys and gnathostomes share the same two WGD events and that these took place before the lamprey-gnathostome divergence.The ancestor of gnathostomes (jawed vertebrates) is generally considered to have undergone two rounds of whole genome duplication (WGD). The timing of these WGD events relative to the divergence of the closest relatives of the gnathostomes, the cyclostomes, has remained contentious. Lampreys and hagfishes are extant cyclostomes whose gene families can shed light on the relationship between the WGDs and the cyclostome-gnathostome divergence. Previously, we have characterized in detail the evolution of the gnathostome corticotropin-releasing hormone (CRH) family and found that its five members arose from two ancestral genes that existed before the WGDs. The two WGDs resulted, after secondary losses, in one triplet consisting of CRH1, CRH2, and UCN1, and one pair consisting of UCN2 and UCN3. All five genes exist in representatives for cartilaginous fishes, ray-finned fishes, and lobe-finned fishes. Differential losses have occurred in some lineages. We present here analyses of CRH-family members in lamprey and hagfish by comparing sequences and gene synteny with gnathostomes. We found five CRH-family genes in each of two lamprey species ( Two aremed UCN2  and UCN3med UCN2 , both ofmed UCN2 , but submed UCN2 .Ciona intestinalis and Ciona savignyi), the cephalochordates (amphioxus Branchiostoma floridae), and ambulacrarians , all have a single CRH-like gene , the spotted gar , and the elephant shark . Gene losses have occurred in some lineages (Diverging scenarios have been proposed to explain the origin and evolution of the CRH family in relation to the emergence of the vertebrates . Lovejoylineages .Petromyzon marinus), one of which is a somatic genome from adult liver and the other a recently assembled germline genome, which is essential because somatic lamprey cells delete much of the genome in adult tissues  and was obtained from mature testis (Eptatretus burgeri)1. Nonetheless, analyses of agnathan gene families and genome segments have been inconclusive regarding the temporal relationship between the two WGD events and the cyclostome-gnathostome divergence, which is why different scenarios have been proposed. Analysis of the somatic sea lamprey genome suggested that the most recent WGD (2R) is likely to have occurred before the divergence of the ancestral lamprey and gnathostome lineages . Our data show that lampreys and gnathostomes have the same number of CRH family members in both of the peptide subfamilies. Furthermore, the lamprey genes are located in gene neighborhoods that resemble those that we have previously reported for gnathostomes, although some rearrangements have taken place. The most parsimonious explanation for these similarities is that lampreys and gnathostomes share five CRH orthologs that arose by chromosome duplications of two ancestral peptide genes. This would suggest that lampreys share the same genome doubling events as gnathostomes, albeit clouded by chromosomal recombination and changes in gene order along the chromosomes.Petromyzon marinus) and four from the Arctic lamprey (Lethenteron camtschaticum), were used to identify the missing genes and the scaffolds for all of the peptide genes in the sea lamprey and Arctic lamprey genome assemblies (available from NCBI database). The predicted mature peptides from lamprey were used to search for homologs in the inshore hagfish (Eptatretus burgeri) genome available from ENSEMBL. The identity of the CRH members that were retrieved was confirmed by submitting to the InterProScan tool2 or by sequence homology.The mature predicted CRH-family members from our previous study , two fro3.The complete deduced precursor sequences for both lamprey and hagfish CRH-family members were retrieved. Mature peptides were predicted by comparing with the gnathostome peptides and by localization in the sequence of putative proteolytic dibasic cleavage sites. Amino acid sequence identities were calculated using the Clustal Omega , availabCiona intestinalis and Ciona savignyi) CRH-like orthologs were used  and human (Homo sapiens) as reference species. The gene environment of the Arctic lamprey homologous genome regions were predicted using a local installation of AUGUSTUS 2.5.5 , a cephalochordate (Branchiostoma floridae), or from the nematode (Caenorhabditis elegans) and fruit-fly (Drosophila melanogaster), and these were used to root the trees. Sequence alignments were performed using the AliView interface with MUSCLE, trees were carried out using the ML implemented in PhyML with automatic selection model, and sequence branching support was given by the Approximate Likelihood-Ratio Test (aLTR). The resulting trees were displayed in FigTree.To better comprehend the evolution of the lamprey and hagfish CRH members, phylogenetic analysis of their neighboring genes families was performed to investigate whether they had undergone similar evolutionary events. Orthologs of lamprey neighboring genes were retrieved from human, chicken, coelacanth, spotted gar, zebrafish, and elephant shark genomes available from ENSEMBL or NCBI. The invertebrate orthologs were retrieved from either two tunicates  and two cephalochordates (B. floridae and B. lanceolatum).To deduce the putative ancestral pre-vertebrate CRH genomic region, we have used all the conserved cyclostome and gnathostome CRH-family neighboring genes to search for homologous genomic regions in invertebrate chordates where a CRH-like peptide gene has been described: two tunicates  to lamprey UCN-a. The absence of additional hagfish CRH-like genes is probably due to incomplete genome assembly.Phylogenetic analysis with the BI method of the agnathan and gnathostome deduced mature peptides  or the cThe five peptides from the two lampreys form five closely clustered pairs reflecting orthology between the species. One hagfish sequence clusters closest to the lamprey UCN_a sequences. The agnathan sequences do not cluster with clear bootstrap support with each of the five gnathostome peptide clades. The agnathan mature peptides and precursor sequences generally tended to radiate earlier and at the base of the discrete gnathostome peptide clades.The deduced mature peptide sequences of the cyclostome CRH-members were aligned and compared with the gnathostome orthologs according to clustering obtained from the phylogenetic tree . Amino aThe S-L-D motif that is conserved at the N-terminus of the gnathostome CRH-members (except in the UCN clade) is also conserved in all agnathan peptides, including the asparagine (R) residue that is localized near the C-terminus and the alanine (A) residue located three positions before. Motifs characteristic of the CRH1/CRH2/UCN1 subfamily , in red The gnathostome CRH-family genes are composed of two exons, the second of which encodes the entire peptide precursor . CharactThe gnathostome CRH family genes map to two distinct paralogons: The CRH1/CRH2/UCN1 genes are located on separate chromosomes in the paralogon that also contains the genes for the opioid receptors  and the The conservation of synteny does not seem to extend to tunicates; despite the existence of a CRH-like peptide in Ciona, no clear homologous genomic region composed by similar genes to those of the vertebrate CRH family members was found. Likewise, no such conserved gene synteny region could be identified in the cephalochordate genome.The three sea lamprey CRH-family members were found in scaffolds 3, 17, and 40, respectively. A total of 21 neighboring gene families that are syntenic with the gnathostome CRH1/CRH2/UCN1 paralogon were identified and characterized . Six of The comparison with the Arctic lamprey  confirmsThe hagfish genome has only a single member of the CRH1/CRH2/UCN1 subfamily . This scPhylogenetic analyses were carried out for all of the 21 neighboring gene families and revealed a similar topology as for the CRH-family genes, i.e., the duplications seem to have taken place in the time range of early vertebrate evolution . OccasioThe lamprey sequences vary more in their positions in the phylogenetic trees. Sometimes they branch off basally to the gnathostome members. Also, the different gene family members from lampreys may group together as if they had been duplicated in this lineage, although the synteny and paralogon analyses support simultaneous duplication along with the gene neighbors in large chromosomal blocks or entire chromosomes.The two sea lamprey UCN2/UCN3-like genes map to scaffolds 57 and 82, respectively. Genes that belong to this paralogon were also found in four other scaffolds . A totalConsidering that there are also several gene pairs present in these chromosomal regions in the various vertebrate genomes, a picture emerges of fourfold symmetry, albeit with weak representation of the fourth member in this paralogon. Overall, these observations point to a paralogon with four members, as with the CRH paralogon, thus most likely reflecting similar genomic events. Like in the other paralogon, many neighboring gene families remain as singletons both in lamprey and in gnathostomes.Comparison of the two lamprey species reveals high similarity of not only gene repertoire, but also gene order . The hagPhylogenetic analyses of all of the neighboring gene families overall display a similar topology as for the CRH/UCN genes. All of the multimember families display gene duplications in the time range of the early vertebrates WGD events . Some ofPetromyzon marinus and the Arctic lamprey Lethenteron camtschaticum, and the hagfish Eptatretus burgerii, have allowed us to analyze scaffolds containing several neighboring genes to shed light on the agnathan-gnathostome divergence in relation to the two WGD events.Our previous analyses of the CRH family allowed us to conclude that five genes arose before the gnathostome radiation as a result of the two WGD events, which triplicated one ancestral gene and duplicated the other . We alsoFive CRH-family genes were found in both of the lamprey species, confirming our previous conclusion  and showOne of the two identified hagfish peptides clustered most closely to lamprey UCN_a , 2, withOne reason that it is difficult to establish orthology between lamprey and gnathostome gene or protein family members may be that lamprey genes are under selection pressure to have high GC content, especially in coding regions . This prAs an additional approach to distinguish orthologs and paralogs, we and others have analyzed the repertoire of neighboring gene families in numerous studies. We therefore analyzed the CRH neighbors and their families in the scaffolds containing the lamprey and hagfish CRH-family genes. As shown in An important result of our analyses of the lamprey scaffolds is that some of the neighboring gene families are quartets: one in the CRH/opioid paralogon (TRIM) and two in the UCN/opsin paralogon (FOXP and LRRN). In addition, several neighboring families are triplets, six in the CRH/opioid paralogon and two in the UCN/opsin paralogon. Two of the lamprey families, RSPO and VSNL1 , actuallCounting also the CRH1/CRH2/UCN1 family, this means that in total 13 out of 37 gene families in these two sets of scaffolds contain gene families consisting of three or four (or five) members in lampreys. In addition, pairs of related genes are present in different combinations of these chromosomal regions, further corroborating relatedness. The most parsimonious explanation for this would be a quartet of related chromosomal regions. By parsimonious reasoning, the simplest explanation would be that lampreys have undergone the same two WGD events as gnathostomes, thus sharing the same 1R and 2R genome doublings. The number of gene families with quartets and triplets of genes is not quite as high as in spotted gar, suggesting that lampreys have lost some family members, as seems to be the case for human and chicken.L. camtschaticum (a.k.a. L. japonicum)  gnathostome regions. For instance, in the UCN/opsin paralogon, there are two UCN genes in the lamprey that might correspond to spotted gar linkage groups LG5 and LG8, both of which have rather complete representation of the neighboring gene families. One of these could correspond to lamprey scaffold 57 and the other to scaffold 82. However, when looking at the gene families that are quartets, namely FOXP and LRRN, neither of these two scaffolds can be complemented by either of the scaffolds 10, 27, or 49, because all three of these contain representatives for both of these quartet families. There are a number of such examples where combination of chromosomes in either lamprey or one of the vertebrates leads to “collision,” as has also been observed for the somatostatin gene regions in ponicum) . One posRecently, three CRH-family members were amplified from the sea lamprey brain cDNA . The thrC. elegans, in several arthropods including the fruit-fly D. melanogaster, and also in annelids, showing that the first gene duplication occurred prior to the protostome-deuterostome divergence  with gnathostomes but also the second WGD event. Some previous analyses of the sea lamprey genome described gene family relationships and conserved syntenic regions consistent with a shared 2R event . HoweverHomologs of the five gnathostome CRH-family members and their neighboring gene families were characterized in the genomes of two lampreys and one hagfish. In lampreys, like in gnathostomes, five members of the CRH family were identified, presumably representing orthologs. However, the exact orthologous relationships could not be resolved even after synteny and paralogon analyses, presumably due to crossing-over events or other rearrangements that have changed their chromosomal positions. Although orthology between lampreys and gnathostomes cannot be determined with certainty, our detailed analyses nevertheless find strong support for lamprey chromosome quadruplication in two paralogons, and thus that lampreys  have undergone duplications that are highly similar to those experienced by gnathostomes. Most parsimoniously, these duplication events were probably the same as in the gnathostomes, i.e., WGD events, and the lamprey-gnathostome divergence took place soon after the second WGD event.The accession numbers of all sequences used in this study were retrieved from public databases and their accession numbers are available in JC, CB, and DL planned the study, evaluated the results, and wrote the manuscript. JC and CB collected the data and performed the analyses. All authors critically read and contributed to improve the manuscript.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.


The increasing incidence of neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease represents a significant burden for patients and national health systems. The conditions are primarily caused by the death of neurons and other neural cell types. One important aim of current stem cell research is to find a way to replace the lost cells. In this perspective, neural stem cells (NSCs) have been considered as a promising tool in the field of regenerative medicine. The behavior of NSCs is modulated by environmental influences, for example hormones, growth factors, cytokines, and extracellular matrix molecules or biomechanics. These factors can be studied by using well-defined hydrogels, which are polymeric networks of synthetic or natural origin with the ability to swell in water. These gels can be modified with a variety of molecules and optimized with regard to their mechanical properties to mimic the natural extracellular environment. In particular modifications applying distinct units such as functional domains and peptides can modulate the development of NSCs with regard to proliferation, differentiation and migration. One well-known peptide sequence that affects the behavior of NSCs is the integrin recognition sequence RGD that has originally been derived from fibronectin. In the present review we provide an overview concerning the applications of modified hydrogels with an emphasis on synthetic hydrogels based on poly(acrylamides), as modified with either cationic moieties or the peptide sequence RGD. This knowledge might be used in tissue engineering and regenerative medicine for the therapy of spinal cord injuries, neurodegenerative diseases and traumata. Recently, the notion emerged that the three-dimensional (3-D) organization of the ECM exerts specific effects , for example in neurospheres and inside the ventricular zone of the developing cortex, which is the location of the proliferating NSCs  or [2-(methacryloyloxy)ethyl]-trimethylammonium chloride, which enhances proliferation, differentiation, and survival of encapsulated NPCs (L-lysine) is superior to gels bearing quaternary ammonium ions and is therefore a promising material for the regeneration of the CNS  on top of a hydrogel film 3-D cell encapsulation has gained significant interest in the past years since 3-D hydrogel constructs may mimic the tissue environment of the cells more accurately . MoreoveOne hydrogel system that has been used for 3-D applications is based on self-assembling peptides (SAP). The components of these hydrogels, for example the RADA16 peptide, may also be modified with peptide sequences like RGD or IKVAV . It coulAn often used hydrogel system are the HA based hydrogels . The HA in situ-gelation inside the injury (While some hydrogels are already used for medical applications such as wound treatments, many challenges have to be overcome for them to be used in patients for different applications. Using human embryonal stem cells for research is associated with ethical problems. In this respect, adult stem cells and induced pluripotent stem cells circumvent these problems and are the main target in clinical trials for hydrogels. Using patient-derived cells reduces immune responses of the body to the hydrogel implant. Furthermore, endogenous stem cells inside the injured tissue may be stimulated by the implanted modified hydrogel to proliferate and differentiate into the required cell types . On the e injury . Dependie injury . The dege injury . In addiin vitro experiments using distinct ECM molecules generate impressive results, but are limited in their abilty to mirror the in vivo situation in an organism. In order to create a more natural environment artificial and natural-derived hydrogels are investigated. Recent advances in materials science have led to innovative biomaterials for use in stem cell research. While in the beginning the research effort was mainly focused on a better understanding of the influencing parameters such as topological cues, biomechanics or bioactive components on neural stem cell behavior in 2-D hydrogel films, there is a clear shift toward the development and application of 3-D cell encapsulation materials that resemble the natural environment of NSCs more closely. Synthetic 3-D hydrogels with multiple functionalities such as peptides or growth factors have been demonstrated to support very efficiently the differentiation of human NSCs into neurons and the formation of neural networks (In summary, the great interest in regenerative medicine to cure neurodegenerative diseases resulted in an expansion on research concerning the development of the nervous system. Soon it became obvious that ECM molecules, like laminins, tenascins, and fibronectins, are important for normal brain development. Single domains and peptides of these molecules have specific functions regarding the behavior of NSCs by activating intracellular signaling pathways via binding to cell surface receptors, like integrins. Nevertheless, networks . TherefoKG, NS, RW, and AF wrote and revised the manuscript. KG and NS designed 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.


A passive wireless impedance-loaded orthogonal frequency-coded (OFC) surface acoustic wave (SAW) sensor for wireless sensor networks was proposed in this paper. One of the chips on OFC SAW tag is connected to an external sensor, which could cause a phase shift in the time response of the corresponding part on the SAW device. The phase shift corresponds to the sensed quantity, which could be temperature, strain, vibration, pressure, etc. The OFC SAW tag is isolated by a proper package from the direct effect of the measurand on the device’s response which could avoid the multiple measurands coupling. The simultaneous work of multiple sensors is guaranteed by orthogonal frequency coding. By processing the response based on an extended matched filter algorithm, sensing information of the specific coded OFC device can be extracted from the superimposed response of multiple independent encoded sensors. Compared to previous methods, the proposed method can produce a more flexible passive (battery-free) wireless sensor suitable for large-scale wireless sensor networks. Simulation and experimental results demonstrate the effectiveness of the sensor. Wireless sensors could be used for structural health monitoring (SHM) systems which are desired on most of the aircraft for dynamic monitoring ,2,3. In A spiral inductive loop antenna with a pressure-sensitive capacitive element could realize wireless passive pressure sensing . The appSurface Acoustic Wave (SAW) sensor is a type of micro-machined passive wireless sensors that have been widely investigated and used for strain, pressure and temperature sensing, etc. ,20,21,22SAW sensors could be classified into two types: delay line SAW sensors which could reflect the measurand by the time delay of the reflectors and SAW resonators (SAWR) which could reflect the measurand by the variation of the resonant frequency . SAW senThe principles for separating the transmitted and received signals include time-domain division (TDD), frequency-domain division (FDD) . HoweverTo expand the capacity of SAW sensors, an OFC SAW sensor based on the principle of orthogonal frequency-coded division is proposed in ,29,30. TA novel impedance-loaded OFC SAW sensor is proposed in this paper. The device identification is accomplished by orthogonal frequency coding, and the measurand is sensed by an external sensor connected to the reflectors on the SAW device. The relationship between the impedance value of the external sensor and the response of the SAW device is investigated, the effect of the external impedance on the orthogonality is simulated, and an extended matched filter algorithm is proposed for the information extraction. Simulation results and experimental results show that the presented impedance-loaded OFC SAW sensor could provide a large capacity for passive wireless sensor networks while avoiding the multiple measurand coupling effects on the SAW device.Orthogonal Frequency Coding is a spread-spectrum coding technique. An OFC signal could have both frequency and time diversity which will provide a systematic way of implementing code in a SAW device embodiment . For an The time-domain response of an OFC signal could be expressed as:According to the orthogonality in orthogonal frequency coding, the product of the local single carrier frequency The local single carrier frequency In Impedance-loaded SAW sensors use electrically loaded IDT to avoid the coupling effect of multiple measurands caused by the direct measurement of the SAW sensor . A classIn  cascade . The refatrix as :4)P11 SAW device has a rather large capacity for multi-sensor operation, the number of available codes could be In this paper, the topology of an impedance-loaded Orthogonal Frequency-Coded SAW sensor is presented. The schematic of the proposed sensor is given in In C, Based on Equation , there iEquation  could beFor the narrowband case, it could be approximated that The impedance-loaded OFC SAW device shown in Since the approximately uniform of the amplitude response for all the five coded reflectors is achieved by apodization which is a cosine weigh to the reflectors, the amplitude response of the total device is quite low, but OFC SAW has an advantage of enhanced signal processing gain which is maintained by the inherent of the spread-spectrum technology. Moreover, the reflectivity of the reflectors could be further improved by embedding the reflector electrodes into the substrate.Based on the orthogonality between different coded SAW device, the response of the specific coded SAW device could be extracted from the superimposition echoed data of the multiple sensors. Since the last chip on the OFC SAW device is slightly deviated from the ideal orthogonal code, the effect of the phase shift value on the orthogonality must be taken into account.The impulse response From The impulse response From The traditional matched filter algorithm is not suitable for the response extraction of the specific coded impedance-loaded OFC SAW sensor from the superimposition echoed data of the multiple sensors, because the phase of the last chip slightly deviates from the ideal orthogonal code. From Equation , the phaThe echoed data reflected from the SAW device Assume the reference signal used for echoed data processing is The correlation function between When Assume A series of impedance-loaded OFC SAW sensors with A comparison of the experimental result and the simulation result of the impulse response for the sensor with OFC code (12345) when external impedance The experimental setup is shown in A series of chirp signals which are transmitted by the interrogator and received by the impedance-loaded OFC SAW sensors are shown in The external impedance is electrically loaded to the SAW sensor with the code (12345). A comparison of experimental and simulation results of the relationship between external impedance values and the phase variations in impedance-loaded OFC SAW sensors is shown in When the external capacitive value is changed from 0pF to 30pF, the relative phase shift of the sensor response which is extracted by the extended matched filter algorithm from the superimposed response of the five sensors will be changed from 0 to 60 degree accordingly.From The previous OFC SAW sensors have large coding capacity for multi-sensors operation while the problem of multiple measurands coupling is hard to resolve ,29,30, sIn this paper, a novel impedance-loaded OFC SAW sensor which is feasible for large-capacity passive wireless sensor networks was presented. A single-value relationship between the phase shift value and the variation of the external impedance is built. The effect of the phase shift value on the orthogonality of OFC coding is analyzed. An extended matched filter algorithm is proposed to extract the phase shift value. Simulation and experimental results demonstrate the effectiveness of this sensor. This impedance-loaded OFC SAW sensor can be used not only for structural health monitoring but also for various physical, chemical and biological measurements by replacing the external sensor. This novel impedance-loaded OFC SAW sensor could provide a large capacity for passive wireless sensor networks.


With the current extensive availability of synthetic-aperture radar (SAR) datasets with high temporal  and spatial resolution , radar remote sensing possesses an increasing potential for the monitoring of glacier surface motion thanks to the nearly weather and time-independent advantages. This paper proposes a robust subpixel frequency-based image correlation method for dense matching and integrates the improved matching into a workflow of glacier surface motion estimation using SAR intensity images with specific pre-processing and post-processing steps. The proposed matching method combines complex edge maps and local upsampling in the frequency domain for subpixel intensity tracking, which ensure the accuracy and robustness of glacier surface motion estimation. Experiments were carried out with TerraSAR-X and Sentinel-1 images covering two glacier areas in pole and alpine regions. The results of the monitoring and investigation of glacier motion validate the feasibility and reliability of the presented motion estimation method based on subpixel gradient correlation. The comparative results using both simulated and real SAR data indicate that the proposed matching method outperforms commonly used correlation-based matching methods in terms of matching accuracy and the ability to obtain correct matches. As an important part of the albedo feedback mechanism of the local and global climate systems, glaciers cover approximately 10% of the Earth’s surface . It is eIn the early stage, the glacier studies depended on compulsory field surveys by glaciologists. To acquire sufficient and reliable measuring data, researchers are obliged to travel to distant glaciers, which are usually located in mountainous areas with highly hazardous environments. However, the collection of measuring field data can be conducted at dozens of positions at specific time points, which greatly depends on the weather condition and the situation of accessibility and takes much time and money. Therefore, obtaining reliable measurement data on a large-scale glacier has long been a difficult task . To addrAn improved robust frequency-based image correlation method, which combines complex edge maps and local upsampling in the frequency domain for subpixel translation estimation, is introduced and integrated into a workflow of glacier surface motion estimation using SAR intensity images.The reliability and feasibility of the presented dense motion estimation method based on subpixel gradient correlation is demonstrated by using TSX and Sentinel-1A (S1A) images covering two glacier areas in pole and alpine regions.With the launch of more advanced SAR satellites, such as TerraSAR-X (TSX) and Sentinel-1, the increasing availability of spaceborne SAR datasets can reach a short temporal baseline of a few or a dozen days and a high spatial resolution in the order of ∼1 m. The high temporal and spatial resolution of SAR data enables the fine-grained and detailed observation of glacier surface motion, and thus offers more potential possibilities and opportunities for glacier displacement monitoring with offset tracking. On the other hand, the high temporal and spatial resolution puts forward higher demand on the accuracy and robustness of offset tracking using SAR intensity images. Subpixel matching method is a decisive component of offset tracking, which largely dominates the final performance of motion estimation. In this study, we develop a robust subpixel frequency-based image correlation method, called locally upsampled gradient correlation (LUGC), to achieve reliable offset tracking of SAR intensity images, and validate the effectiveness in the applications of glacier surface motion estimation. Our contributions that are specific to monitoring the glacier surface motion include:The optical images acquired by Earth observation satellites are the most commonly used datasets for glacier monitoring. However, the frequent cloud cover and sunlight variation in polar and high mountain areas inevitably make the permanent collection of optical data impossible, which leads to insufficient data coverage . ContrarInterferometric methods are regarded as an accurate technique for glacier monitoring, with the highest sensitivity to surface motion. With the aid of the subsequent phase changes or the sequence of transmitted wavelengths, it is possible to accurately measure the movement of surface feature patterns ,11. DiffOffset tracking-based methods usually use the amplitude information of SAR data, which is mainly carried out by image correlation for measuring offsets of points or features on glacier surfaces. Here, feature tracking and intensity tracking are two commonly used methods for offset estimation.Feature tracking relies on the detection of continuous, co-registered image centering on the movement of identifiable objects  or feature points on the surface of a glacier, which can derive a two-dimensional velocity field. Feature tracking methods can be achieved by either manual selection or automatic tracking. In the early days, feature tracking was mainly performed manually , and curIntensity tracking is commonly based on image correlation methods that calculate the similarity between small sliding templates to obtain the relative displacements between image pairs in the distance and azimuth directions, respectively. This technology was first introduced into the field of optical data  and was Similar to the conventional scheme of Earth surface dynamics monitoring from optical images ,37,38, thttp://www.sarmap.ch/wp/index.php/software/sarscape/) following the radar equation principle, speckle noise filtering with an adaptive Lee filter 29], PC wThe different image matching methods were assessed based on two criteria similar to : matchinThe template size plays a crucial role in correlation-based image matching. The selection of optimal template size should be large enough to ensure sufficient textural information for matching, but small enough to decrease the possibility of deformations in the template . In thisx- and y-direction with a sinusoidal transform were applied to model the nonlinear distortions. Here, the period of sinusoidal deformation was set as 900 pixels, and the amplitude was eight and six pixels in the x- and y-direction, respectively.The proposed method and other matching methods were firstly applied to the simulated SAR data to investigate the properties and matching accuracy in a controlled environment. Taking a subset of either TSX or S1A image over glacierized region as the master image of synthetic image pair see a, the slx-direction errors of all the methods are higher than the y-direction ones. The rankings of matching methods in terms of mean absolute error and RMSE are the same. The LUGC method obtains the best matching results and the PEF method the worst results both when it comes to mean absolute error and RMSE. PC-based methods  generate more mismatches than the others, possibly due to the sensitivity of PC to local deformations in cases of a small template size [The results of different subpixel matching methods on these two synthetic image pairs in terms of mean absolute error and RMSE are given in ate size . On the x-direction and y-direction displacement maps of the Taku Glacier derived from the proposed LUGC method with a 64×64 template size. It is obvious that there exist mismatches in the matching results. These noisy estimates are mainly located in low-textured regions, such as snow-covered surfaces and shadowing on the glacier tongue, wherein adequate useful information to determine the correspondence between templates cannot be provided.After the pre-processing of TSX intensity images, the dense matching step was conducted between the orthorectified image pair to obtain the 2D displacements of the glacier surface. x-direction and y-direction displacement maps in both glacierized and stable regions. As can be seen from the figures, most of the mismatches with abnormal values, inconsistent neighborhood, and isolated distribution are automatically eliminated by the post-processing step. The basic trends of the 2D surface motion for the Taku Glacier can be reflected after the outlier removal. The main trend of the glacier flow is from the northwest to the southeast direction, as the Taku Glacier is a tidewater glacier and the ice mass is flowing directly to the river. The surface displacements in the y-direction are relatively consistent downward, while the surface displacements in the x-direction show some local variations. Specifically, the flow direction in the middle part of the study area is reversed, probably because of the branch of Taku Glacier in the right part.The post-processing step considering the displacement range, neighborhood, and connectivity was performed to filter out these erroneous measurements. In x-direction and y-direction displacement maps of the Pine Island Glacier output by the dense matching step and the corresponding filtered displacement maps output by the post-processing step are presented in Using the pre-processed multitemporal S1A intensity images, the Similarly, the magnitudes of surface motions over the glacierized regions were composited using the displacement maps and a manual labeled mask, as shown in The displacement maps of the Taku Glacier and Pine Island Glacier were calculated and filtered using different matching methods and the same pre-processing and post-processing workflow. The numbers of assumed correct matches that passed the filtering operation both over the glacierized regions and over the entire image were counted. The percentages of correct matches for different matching methods are given in It is obvious that the results vary both from region-to-region and from method-to-method. The percentages of correct matches in Taku Glacier are higher than ones in Pine Island Glacier for all matching methods. This is because that Pine Island Glacier has more snow-covered areas especially in the surrounding regions. The textureless areas easily generate noisy matching measurements, which are removed by the filtering operation.Among the compared matching methods, the proposed LUGC method generally outperforms other matching methods and obtains the best performance of the assumed corrected matches after the same filtering operation in all cases regardless of the test regions and template sizes. The higher percentages of correct matches indicate that the LUGC method is able to provide stable and consistent results of glacier motion estimation. The robust performance is attributed to the joint use of the magnitude and orientation information of edge responses and frequency-based local upsampling, which ensure image correlation with salient structural frequency components and efficient subpixel calculation. In contrast, the PC-based methods perform the worst. The PEF method obtains the lowest percentages of correct matches, and the COSI-Corr-F method can only generate reasonable results for Taku Glacier with an initialization using a larger template. This agrees with the situation in the simulated test, but is opposed to the situation in , which fIn addition, it is worth noting that the results for Taku Glacier derived using template sizes of 64 pixels are largely improved compared to those using template sizes of 32 pixels. This demonstrates the significance and difficulty of selecting optimal template sizes. However, the requirements of template size to obtain valid results are diverse for different grid positions, since a small template size is adequate for certain positions. Therefore, an adaptive strategy for adjusting template size based on local structural statistics is highlighted ,64. FutuTo show the performance of different methods in the key part of the glacier surface, each profile was selected in Taku Glacier and Pine Island Glacier as shown in This paper presents a motion estimation method of glacier surface using repeated SAR intensity images based on subpixel gradient correlation. The overall workflow of glacier motion estimation includes pre-processing, dense matching, and post-processing steps. A robust frequency-based image correlation method is developed by locally upsampling the correlation function calculated from the complex representations of image gradients in the frequency domain, which can effectively achieve subpixel dense matching of SAR intensity images. The proposed method is validated using simulated and real SAR data. The monitoring of Taku Glacier and Pine Island Glacier between revisit periods of SAR data is successfully conducted with the motion obtained and the trend investigated. The comparison with commonly used correlation-based matching methods demonstrates that the proposed LUGC method is able to achieve higher matching accuracy, as well as generate higher percentages of correct matches and more stable and smoother motion results. In future work, the presented method will be extended to more detailed applications with multi-pass SAR intensity data, and the improvement of the matching method will be investigated by combining adaptive template strategies and considering better robustness against SAR speckle noises.


Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identified very rapidly to save a patient’s life. This review outlines the main techniques of visualization of rare objects in the blood flow, methods for extraction of such objects from the blood flow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods. The problem of detection and extraction of rare objects from the blood flow arises in a number of situations. This includes the search for the very rare circulating tumor cells (CTCs) at early stages of cancer development by liquid biopsy ,2, the dAccording to the statistics of the World Health Organization for 2016, around 71% of the overall 57 million deaths are caused by noncommunicable diseases, including cardiovascular disease—31% and cancer—16%  and the Babesia microti, B. divergens -, perchlorate  and 1,1’-dioctadecyl-3,3,3’3’-tetramethylindocarbocyanine perchlorate (DiI). The fluorescence of these dyes is greatly enhanced after incorporation into membranes or binding to lipophilic biomolecules weakly fluorescent in water, such as proteins. Along with it, dialkylcarbocyanine derivatives perform high molar extinction coefficients, polarity-dependent fluorescence, and short excited-state lifetime (~1 nanosecond). This is an advantage for flow cytometry applications because it allows for more excitation/emission cycles during flow transit. Additionally, dialkylcarbocyanines are quite photostable and provide a suitable imaging tool for flow cytometry study of live cells due to the distinct fluorescence colors. In particular, DiO emits green light (abs. 484 nm/em. 501 nm), DiI emits orange light (abs. 549 nm/em. 565 nm), DiD emits red light (abs. 644 nm/em. 663 nm), and finally, DiR emits deep red light (abs. 748 nm/em. 780 nm). Among them, DiO can be effectively excited by 488 nm argon laser, and its fluorescence spectrum is comparable with that of FITC, whereas the 633 nm He-Ne laser is convenient for DiD. TRICT and DiI can be visualized by the same optical filter sets due to their excitation/emission wavelengths similarity. Lipophilic carbocyanines as membrane-bound counterstains have been appreciated for their use to visualize membrane fusion and cell permeabilization, for cell migration and proliferation studies ,118.One more approach to distinguish cells of interest is to make the cells fluorescent themselves without external labeling that might potentially affect their behavior. This is achievable through the modern molecular genetic methods allowing for the transfer of fluorescent protein (FP) genes into the cells. The FP labeling for bioimaging is one of the most widespread applications of FPs. The general strategy for cell labeling is to include the nucleotide sequence of FP responsible for fluorescence in the gene tailor the gene body of the labeled cell protein depending on its structure. In some cases, a flexible Gly-rich linker between the FP and the labeled protein is used in order to prevent potential steric conflicts . The mosAequorea Victoria . . 156]. TSince superparamagnetic nanoparticles have a natural magnetic polarization, a non-uniform magnetic field (or a magnetic field gradient) is required to move them by magnetophoretic force analogous to the dielectrophoretic separation. Thus, all magnetic separation methods employ the structures creating local inhomogeneity of the magnetic field acting as trapping or deflecting sites for the cells labeled with magnetic particles. This was successfully realized by Miltenyi et al., who first proposed in 1990 a parallel cell separation technique for routine use in laboratory currently known as a conventional magnetic-activated cell separation or MACS . They usZborowski’s group has suggested an alternative way for continuous flow magnetic separation developing a quadrupole magnetic flow sorting approach (QMS) ,227. GenThe main benefit of the quadrupole field is the fact that magnetophoretic force acting on the particle is finely defined by the particle position over the channel cross-section. The QMS separation concept is based on the cell magnetophoretic mobility that is the velocity of the cell per unit of the magnetic force. Passing through the channel, the cells labeled with magnetic nanoparticles are deflected to the channel walls, whereas less mobile unlabeled cells remain near the core rod forming two outflows separated by a flow splitter. The inlet flow of the initial cell mixture is also separated from the channel wall by the flow splitter to avoid the non-labeled cells pass near the magnet and come to the positive outflow with the magnetically isolated cells. To date, QMS has been successfully employed for positive selection of magnetically labeled CD34+ blood progenitor cells from blood circulation ; depletiHowever, modern tendencies in magnetic cell separation are mostly related to the development of microfluidic sorting systems. The employment of the microfluidics allows for the relatively simple generation of local high gradient magnetic fields in the desired area of the microchip and preparation of microfluidic channels of various shape and geometry by well-established microfabrication techniques. Additionally, microfluidic systems significantly reduce the required volume of the analyzed sample. Novel microfluidic platforms for magnetic cell separation are constantly developed, numerically simulated, and tested for continuous-flow separation of magnetic beads An important aspect of magnetic cell sorting is the isolation of the cells with an inherent magnetic moment. There are several reasons to exploit the label-free separation of intrinsically magnetic cells rather than utilize labeling approaches. One of them is purifying biological samples from an excess of RBCs for diagnostic or therapeutic applications. For instance, isolation of RBCs form stem cells is desirable at bone marrow transplantation . AdditioPlasmodium parasites affecting host RBCs. During the life cycle, the parasites metabolize most of the cellular hemoglobin to hemozoin exhibiting paramagnetic properties in contrast to oxygenated hemoglobin, which is diamagnetic [Furthermore, the development of continuous flow magnetic isolation of infected RBCs is of great importance with respect to malaria treatment. Malaria is an infectious disease caused by magnetic . Moreovemagnetic . This dimagnetic . Moore emagnetic . Furthermagnetic . They remagnetic ,265,266,magnetic  They desmagnetic .Despite the current popularity of microfluidic approaches for magnetic cell separation, these methods have a considerable drawback that is a small volume of processing blood samples and thus the separation of rare circulating objects . Although microfluidic isolation can almost completely remove the target objects (with efficiency about 90%–95%), a typical sample volume for microfluidic investigation is about 5–10 mL, and this can only contain a tiny amount of rare blood biomarkers that is insufficient for adequate analysis and reliable diagnosing . TherefoFinally, we refer to some recent reviews covering various issues related to magnetic cell sorting. Plouffe et al. have reviewed the application of magnetic particles for cell isolation . This inAcoustophoresis is an application of the acoustic field for cell sorting see c. AcoustUltrasound waves used for acoustophoresis can be divided into standing and traveling waves . In turnThe standing acoustic waves occur under resonance conditions. To perform acoustophoretic sorting with standing BAW, ultrasound is typically applied transversely to the cell flow direction. The standing BAWs will occur in a microfluidic channel if the ultrasound wavelength matches the channel width . This kiAt present day, there are microfluidic devices employing both standing and traveling surface acoustic waves for the selection of cancer cells from the blood. This multi-stage sorter used standing waves for cell focusing in the central part of the channel and traveling waves for displacing of target cells to the desired outlet .The sorting approaches described above include active particle or cell separation techniques. There are also passive methods developed for the separation, isolation, or enrichment of particles and cell populations based on typical differences in their size, shape, or morphological parameters. Inertial forces, hydrodynamic propagation, and deterministic lateral displacement are applied to sort different objects ,296.One of the commonly used approaches for label-free passive cell separation is inertial focusing in microfluidic channels. The theoretical background and detailed description of the separation process were summarized by Di Carlo . BrieflyThe curved microchannels may be employed to produce inertial forces. Considering the laminar inertial flow, the liquid flowing through the curved channel results in the re-circulating secondary flows that are formed due to a mismatch of the liquid velocity in the channel center and near-wall regions b. These As recent practical examples, Nathamgari et al. used curved microchannel to isolate single stem cells from cell clusters after dissociation from the initial tissue . Son et Staphylococcus aureus bacteria exploiting this approach [However, the preparation of curved and wave-shaped microchannels might be challenging, and, for some applications, the straight microchannels are preferable due to the ease of preparation and parallelization. Thus, one of the possible approaches to improve inertial separation with straight channels is the employment of the channels with an asymmetrical cross-section. For instance, Moloudi et al. investigated the possibility of particle separation in a trapezoidal straight microchannel . They fiapproach .eff) is more than a critical radius (Rc), the particle moving around the post will be deflected to the neighbor streamline [C remain in the same streamline and keep following a “zigzag” but eventually straight path . DLD is a method that utilizes an array of posts placed in the microchannel in a defined manner that is every following raw is shifted latterly toward the previous one at a set distance . This lereamline . This acpath see a. The crT. cyclops parasites from RBCs in whole blood [First, DLD was introduced as a separation technique by Huang et al.  They demle blood , and carle blood . Furtherle blood . They file blood . Previoule blood . Howeverle blood . The clole blood . This dele blood . WithoutAnother issue is that DLD, as other microfluidic passive separation approaches, implies a laminar fluid flow with Re less than 1. This is the pre-requisite for the formation of the defined laminar streamlines and a predictable movement of the particles through the post array. This results in a relatively low fluid flow rate and restricts the separation performance. On the other hand, the increase of the fluid velocity at high flow rates leads to the domination of inertial forces that alter the streamlines, which resulted in the formation of the vortices in the wake of the posts. The vortices increase the hydrodynamic radius of the posts reducing the critical radius of the particles. Recently, Dincau et al. reported on the numerical simulation and experimental evaluation of the performance of the DLD device operating in the high-Re mode (10 < Re < 60) . They fiAdditionally, DLD may be employed for separation of particles and cells by their shape involving two types of phenomena: differences in length scale and differences in transport properties . For insAlthough the design of DLD devices still is under consideration for every particular application, they were shown to be effective for cell isolation for cancer diagnostics. The examples include but not are limited to the works on CTC isolation mentioned above. Additionally, Wunsch et al. demonstrated the approach for the preparation of a nano-scaled DLD array that is suitable for the separation of exosomes . More reTo conclude this subsection, we refer to the review of McGrath et al. devoted to the DLD separation . This rePinched flow fractionation (PFF) is an approach for the separation of particles according to their sizes in laminar flow . It was Yamada M. et al. separated polystyrene microspheres with a diameter of 15 μm from 30 μm ones . The autAlternatively, the separation resolution was enhanced by a simple geometric modification of the primary PFF design like adding a snakelike part to channel . This reThe first step towards nano-scale separation was realized by Y. Sai et al. by separation of 0.5 μm from 0.86 μm polystyrene microspheres . They suIn concentrated samples, interactions between particles increased, which leads to decreasing the selectivity of the PFF system. Thus, the influence of concentration needs to be investigated. The quantitative influence of the pinching intensity in the balance between the requirements of selectivity and minimal dilution was discussed . As a reEventually, the PFF technology was extended to a passive hydrodynamic cell sorting for leukocyte enrichment . In addiFurther PFF improvements can be developed by using other separation techniques in combination with PFF, for instance, sedimentation , centrifAfter the flow cytometry data is collected, a detailed analysis should be performed. At the beginning of flow cytometry development, the analysis was carried out manually  and, theConventional flow cytometry methods provide an intrinsic set of features describing single objects in the flow. It usually includes forward scatter and side scatter channels along with signals from PMTs detecting fluorescence in a number of wavelength bands. Imaging flow cytometry, however, requires conversion from raw images of single objects into a set of features that allow discriminating properly different object classes. Feature construction and selection of separation criteria for a long time was a complex procedure that requires high qualification and a lot of expertise from the device operator. Progress in flow cytometry increased both the number of cells in the experiment and the amount of information obtained from single cells, so the processing methods became more complex. On the other hand, modern progress in machine learning and deep learning methods start to make formerly complex procedures completely automatic and less erroneous in general.In the conventional analysis of flow cytometry data, expert works with a set of features—the values that describe each object in the flow. The most basic is the intensity of fluorescence in different spectral channels along with front scatter and side scatter light intensities. The multidimensional space of features is usually orthogonalized by a compensation matrix that describes how to minimize the crosstalk between channels. Afterward, the feature space can be distorted by some transfer function  that provides cut-off for the outliers or compression of the rare high-intensity events into the space near the major data points. The essential part of the data processing is based on gating. The features that provide separation of object subpopulations are selected and plotted as 1D or 2D plots. Then the data points in a range (for 1D) or a region (for 2D) of values are selected manually on the plot by visually monitoring the points clusters and the data for objects subpopulation is extracted, so the next separation can be done on this subset.To perform data analysis after receiving the signals, specific features should be determined. The combination of these features should characterize the cells of interest in the most accurate way. Until recently, classical computer vision (CV) and machine learning (ML) algorithms, such as thresholding (for feature extraction) or gradient busting and random forest (to solve classification problems) were mainly employed in the analysis of flow cytometry data . HoweverA lot of effort was made to define the automatic gating procedure for flow cytometry data ,363,364 Spanning-tree Progression Analysis of Density-normalized events (SPADE)  organizeFlowSOM  is anothThe third automatic approach for the detection of subpopulations from a flow cytometry dataset is based on the t-distributed Stochastic Neighbour Embedding (t-SNE)  method. Information about automatic methods not using dating summarized in a number of comprehensive reviews, that include Aghaeepour et al.  and MairThe next major step in the automation of cytometry data processing is based on transfer from manual feature construction to automatic. This process in computer science is heavily based on the transfer from classical machine learning algorithms, which separate the data by manually selected and thoroughly checked set of features, to learning approaches, which construct features by learning.Most of the deep learning approaches were based initially on learning convolutions in the separate layers of neural networks (NNs) architecture. However, nowadays there are various and versatile sets of network architectures providing required functionality and the progress in inventing new ones is very fast. First successful approach to construct deep learning models for computer vision was mostly based on a deep network with a lot of layers in the structure forming a pairs from convolution layer that learns transfer methods to construct a features at each level of abstraction and fully connected layers keeping information as a set of weights of selected features . The morAny machine learning model still requires a dataset to be trained on. During the training, deep learning algorithms independently selects geometric features from the available data , followed by the derivation of abstract features from, for example, presence of a vacuole in the image or damage of cell nucleus . As a reDeep learning approach allows us to solve some complex computer vision problems like semantic segmentation (separation of closely packed objects) or object detection with the accuracy of well-trained human experts and sometimes even better, however, it is achieved by the large size of required datasets. The usual balanced set of objects to train a deep neural network for computer vision problems should consist of tens of thousands of objects from each class. Sometimes it can be achieved by artificial augmentation of smaller datasets containing only thousands or even hundreds of objects, but it is mostly a required minimum to train a good working model. The initial marking of the dataset should be done by human experts and it is among the most tedious and complex tasks in a deep learning approach.Additionally, a serious obstacle using deep learning methods in diagnostics may be their interpretability . One or Recently, there were a number of publications describing the complex system providing cell sorting by a combination of all the above-mentioned approaches. Nitta et al. demonstrated an integrated approach to the problem of finding the right cells and sorting them in a real-time combing traditional and deep machine learning . Since nThis paper also demonstrated a well-developed approach to building the entire computing architecture. Often this issue is neglected in research, giving preference directly to data analysis. However, an emerging trend towards the application of flow cytometers in diagnostics, engineering issues will have to be paid more attention. Properly built and optimized computing architecture can significantly increase effectiveness without the need for expensive equipment. In turn, this will make promising flow cytometer-based devices more affordable.Li et al. addressed the problem of imaging cells without fluorescent agents to avoid their impact on cell behavior . The autTanhaemami et al. predicted the lipid content in microalgae cells after nitrogen starvation by application of linear regression and a genetic algorithm (without the use of fluorescent labels) . EulenbeCertainty modern machine learning methods will gradually supersede traditional methods in the processing and analysis of flow cytometry data in the long run. ML and DL proved to be powerful and well-suited tools in cytometry. Probably, quite a few tasks can be solved by purely engineering approaches by adapting existing algorithms. Nevertheless, there is still the problem of further increasing the rate and biological interpretation of the results.Flow cytometry technology has undergone many changes since its invention in the 50s of the last century. The development of new hardware and software for flow cytometry systems as well as fluorophores development expanded the number of science domains in which this technology could be applied. At the dawn of flow cytometry technology, it was used just to count and estimate the size of particles . CurrentThe current state-of-the-art in fluorescent flow cytometry allows to estimate up to 20 independent parameters for each cell and possesses a high throughput possibility—thousands of cells per second . However8 cell/mL for Amnis, 3 × 107 cell/mL for Bio-Rad, 7.5 × 106 cell/mL for Beckman). However, a more detailed analysis of certain cell populations selected during flow cytometry measurement, for instance, genetic analysis requires some post factum procedure. With this regard, the demand for separation of the sample or its return for further analysis arises. Some devices such as Sony SH800, Bio-Rad S3e cell sorters, or Beckman MoFlo XDP High-Speed Cell Sorter have this ability, but this is the exception rather than the rule.Additionally, conventional fluorescent flow cytometry systems do not provide bright field and fluorescent images of events that constrain the ability of real-time verification. Imaging flow cytometry systems successfully solved this problem by combining the statistical power of conventional flow cytometry with the advantages of fluorescence microscopy . The comThe progress in the design of modern flow cytometry devices is strongly related to advances in adjacent areas of research and development. Considering the hardware part, great progress was made in the design of advanced optical systems like structured light illumination schemes, which allow for capturing better quality images on higher speeds of flow with less laser-induced damage of fluorophores. Additionally, the novel optical systems require fewer efforts in precise focusing of detected objects in flow and lens objective on the common spot. Great progress was also made in nonconventional detection methods including flow cytometry approaches for detection in vivo based on photoacoustic and photothermal phenomena. However, these substantial areas are out of the scope of this review.Further, a semiconductor industry constantly provides better detectors that require less light to detect a signal. The modern techniques of image processing are able to take composite digital images by merging processed low-quality signals with properly removed noise from many sensors simultaneously. Semiconductor laser diodes and detectors for the less common part of the optical spectrum starts to be more available in all means and easier to integrate into new device designs.Enormous progress in flow cytometry data processing software is achieved with the development of modern machine learning and deep learning techniques for all stages of data processing. The denoising and sensor fusion, solving computer vision and object detection tasks, grouping objects by the similarity of features that are automatically selected and generated are used everywhere now. All device and processing software vendors are starting now to provide this kind of solution to end-users.With all this combination of novel techniques, the flow cytometry area of research is developing rapidly providing better devices that have better performance and much easier to operate. It pushes flow cytometry towards widely adopted endpoint-of-care devices from the research lab.


To decipher the molecular basis of health and disease, profiling multiple molecular modalities is a common practice . Most of these molecular assays generate lists of genomic loci that are relevant to the trait/phenotype under investigation. Functional interpretation of these lists is often carried out through colocalization and enrichment analyses . Specifically, the authors summarized single nucleotide polymorphism (SNP) enrichment analysis and statistical colocalization analysis. SNP enrichment analysis is one way to identify the tissue/cell types that are relevant for a disease by integrating either genome-wide-significant or a full set of assayed SNPs with molecular annotation tracks . Once the relevant tissue/cell types are identified, further refined analysis using similar statistical analysis methods could disentangle the enrichments in highly similar cell types . Statistical colocalization analysis is one way to interpret novel GWAS findings by linking GWAS findings with likely target genes. This can be achieved by integrating GWAS signal with eQTL data to evaluate whether the same variant is causal in both GWAS and eQTL studies. In addition to summarizing the knowledge and strategies of the SNP enrichment and colocalization analysis, the authors have also provided perspectives on how the state-of-the-art technologies  could be utilized in the future for the interpretation of GWAS findings.Lee et al.) complements the current state-of-the-art methods to aid the functional interpretation of non-coding regions by extending the methodology to not only test the proximity to TSS, but also to enhancers. The authors show that ProxReg provides additional insights into the regulatory mechanisms and binding tendencies of transcription factors .One of the applications of colocalization analysis is for the interpretation of the functions of non-coding genomic regions. GREAT  published in this special issue takes a step further in this direction, and provides large collections of curated genomic tracks . The curated data span across transcriptional regulators, histone modifications, chromatin accessibility, transcriptional events, and chromatin segmentation data.One of the arduous tasks when using colocalization analysis tools to test/generate hypotheses is the need to carefully curate a collection of reference genomic tracks that are annotated thoroughly. Existing tools provide carefully curated collections of reference track collections  with them. Often, subsequent enrichment analyses are carried out on reference genome annotations that are overlapping the LD blocks. LD-annot provides a convenient wrapper around the popular PLINK tool  and compare them across different conditions. One of the main challenges for TADcompare (as noted by the authors) was that no ground truth exists for boundaries of interacting domains, making it difficult to quantify the identified boundaries' biological relevance. To tackle this challenge, the authors of TADcompare used a range of colocalization analyses of epigenomic annotations and also a colocalization-based gene ontology enrichment analysis to determine whether the known genomic features that are characteristic of interacting domains and boundaries are enriched proximal to the identified boundaries and if that is different than background (non-boundaries).The study by Ronzio et al. presents a new pipeline based on colocalization/enrichment analyses to identify regulatory modules of transcription factors (TFs) and TF recruitment rules. Instead of requiring overlap between a pair of ChIP-seq tracks, a proximity-based test statistic is suggested to quantify colocalization. The significance  is computed according to either hypergeometric or Poisson distribution. One possibility in the pipeline is to convert the p-values into scores and perform clustering analysis between the scores of multiple experiments to visualize potential regulatory modules. Further, motif enrichment analysis either relative to the whole accessible DNA or selected windows  could be performed for a pair of TFs. One could draw inferences on the recruitment patterns based on the observed pattern of motif enrichment (motifs for both TFs enriched or only one of them or none). Construction of the background sets in both colocalization analysis and motif enrichment analysis using alternative definitions  would allow one to identify specific regulatory modules.The study by Tan et al. extends the traditional GSEA approach to establish associations between chemical-associated gene sets and gene expression in colorectal and rectal cancers. In the absence of a reliable tool to simulate CNVs from whole-exome sequencing data, Xing et al. developed the SECNVs tool which can generate CNVs with multiple customizable parameter options to mimic realistic CNVs from experimental data. The simulated CNV datasets could be utilized to explore the patterns of enrichment of CNVs in various contexts. For example, earlier others (Alexandrov et al., The study by Overall, this Research Topic summarizes and showcases some of the existing and novel ways of utilizing genome colocalization/enrichment analyses to study a wide range of genetics and genomic research questions. The methods and tools published in this Research Topic extend the state of the art and enhance user convenience in performing genomic colocalization/enrichment analysis. With the continuous increase in the generation of genomic/epigenomic datasets, the interpretation of the resulting genomic regions becomes vital; we expect that the methodological principles of genomic colocalization/enrichment analysis will be utilized in many innovative ways in the future to further aid the functional interpretation of genomics datasets.CK wrote the manuscript. GS, EH, SD, and RL edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adrenocortical carcinoma (ACC) is an extremely rare disease, the incidence of which is 0.7–2 per million population per year in Western countries . The manThis Special Issue comprises 22 papers  presented by international leaders in the field of ACC. The four review papers focus on topics of great interest. Two of these refer to ACC genomics and provide a complete overview of the potential role of epigenetic alterations  in the pAmong the 18 original articles, 7 refer to prognostic parameters and 1 has been previously mentioned. Additionally, another paper assumed the role of investigating the effect of mitotane serum levels on patient prognosis . This paThree papers deal with the genetics of ACC. In one of these, it is shown that the prevalence of altered mismatch repair (MMR) genes among pediatric patients is higher than that in colorectal and endometrial cancer cohorts . The secEcteinascidia turbinata against ACC cells. In addition, the synergistic cytotoxic activity of trabectedin with mitotane provides the rationale for testing this combination in a clinical study [As regards the three preclinical published studies, one of them explored the effect of crosstalk between cancer cells and adipose precursors in the stroma using a co-culture in vitro model. The results suggest that adipose cell precursors could promote cancer cell reprogramming and invasion, opening new perspectives on the mechanisms of ACC aggressiveness and the development of effective therapeutic approaches . The secal study . The thial study .Three clinical papers evaluated the efficacy of therapeutic strategies. Etoposide, doxorubicin, and cisplatin plus oral mitotane (EDP-M)  represenFinally, another paper addresses the issue of whether the hypothalamic–pituitary–adrenal (HPA) axis can recover after cessation of adjuvant mitotane therapy for ACC. Data from 23 patients with pathologically proven ACC, treated with adjuvant mitotane for a minimum of two years, were analyzed, and the results showed that a high proportion of patients achieved HPA axis recovery following cessation of mitotane adjuvant therapy . HoweverIn conclusion, this Special Issue presents some significant progress made in fields related to the genetic and pathophysiology of ACC and outlines some of the advances in the treatment and prognostication of ACC patients. Above all, this Special Issue highlights how bringing together researchers and clinicians from around the world is key to better understanding this challenging disease and improving the available therapeutic strategies.


Drosophila embryos with a focus on E-cadherin dynamics in linking cell and tissue polarization and tissue-scale shape changes.Planar cell polarity and anisotropic cell behavior play critical roles in large-scale epithelial morphogenesis, homeostasis, wound repair, and regeneration. Cell–Cell communication and mechano-transduction in the second to minute scale mediated by E-cadherin complexes play a central role in the coordination and self-organization of cellular activities, such as junction dynamics, cell shape changes, and cell rearrangement. Here we review the current understanding in the interplay of cell polarity and cell dynamics during body axis elongation and dorsal closure in  Epithelia constitute the surface of organs in multicellular organisms and the units of many morphogenetic processes. Epithelial cells adhere to one another to form two-dimensional sheets and constitute permeability barriers for compartmentalization of the body, which is essential for the physiology and protection of the organs and even the whole organisms. Despite their physical integrity and stability, epithelial sheets are intrinsically dynamic and able to restructure in a time scale as fast as minutes . During Planar polarity is based on molecular asymmetries within the epithelial sheet and cells and impinges on the cellular activities leading to tissue-scale shape changes. Cell junctions are at the center of the transition from cells to tissue. The mechanical link between is constituted by adherens junctions with E-cadherin (E-cad)–catenin complexes as the central component. Together with numerous associated proteins varying between cell types and developmental stages, the E-cad complex provides a mechanical link between the actomyosin networks of adjacent cells and coordinates their activities via mechanotransduction .Drosophila embryogenesis, i.e., germband extension and dorsal closure. With these two case studies, we will discuss how cell and tissue polarization are coordinated to give rise to tissue-scale changes in visible morphology.In this review, we will focus on recent progress in two processes of Drosophila development is initiated when the zygotic genome is activated at the transition from syncytial to cellular morphology , known as Shotgun (Shg) in Drosophila, was identified as Armadillo (β-catenin) associated glycoprotein , which initiates DE-cad endocytosis leading to depletion of α-catenin  and two regulatory light chains (RLC) . The Rho-catenin  and Baz/-catenin .runt, eve, and paired imposes a planar polarity on the tissue, which guides the orientation of T1 transitions and thus the directionality of cell intercalation. AP patterning of Drosophila embryo is controlled by a hierarchical genetic cascade starting with localized maternal determinants to the zygotic gap, pair-rule, and segment polarity genes (The initial signal for polarization is provided by the striped expression of anteroposterior patterning genes . The strty genes . The linty genes . The staty genes . Heteropty genes . The molty genes . The Tolty genes . Althougty genes , neitherty genes .Drosophila embryogenesis (Dorsal closure is another prominent morphogenetic process in ogenesis  (Hayes aogenesis . Dorsal ogenesis . Within ogenesis . The mecogenesis , we willThe interface between the two tissues plays an important role. The dorsal-most epidermal cells, the leading edge cells, polarize by an accumulation of F-actin at the interface between their dorsal edge and the amnioserosa interface, which generates a prominent and contractile F-actin cable  Young e. MeanwhiZasp52 mutants embryos, which lack any actin cable but undergo an apparently normal dorsal closure (Zasp52 mutants. These observations suggest that the elongation of epidermal cells is not only due to pulling by the actin cable.Tissue restricted Myo-II depletion in the amnioserosa or surrounding epidermis revealed that the Myo-II dependent contractions within the amnioserosa tissue but not actin cable are required for dorsal closure . However closure . Interes-catenin mutant embryos, in which the actin-binding domain was specifically deleted (Although the amnioserosa cells behave isotropically with respect to their oscillations, the cell junctions at the interface are polarized as seen not only by the actin cable but also by the junction and junction-associated proteins. The epidermis connects with amnioserosa cells via E-cad and integrin-mediated adhesions . Reduced deleted . Further deleted . The Aju deleted . This pr deleted . These sWithin the lateral epidermis during gastrulation, the AP patterning system establishes a system of planar cell polarity, which polarizes junctional and cytoskeletal dynamics and subsequently directs cell rearrangement for the tissue-scale changes in morphology. The finding that members of the Toll-family of membrane receptors are involved in the polarization of the tissue has started to open the black box of molecular links between the transcriptional patterning machinery for axis formation and the cell biological machinery of contractile actomyosin clusters and cell adhesion complexes . E-cad aDrosophila embryos provide a suitable and highly tractable system to study such questions in vivo.Yet missing is an integrative systems-type analysis involving mechanisms of coordination among the direct neighbors but also long-ranging influences to second and third neighbors. Analysis of the temporal and spatial coordination of the identified contractile and adhesive activities will be needed for the step from understanding the individual events such as a junction collapse to the tissue-scale shape changes during morphogenesis. Beyond the individual tissue, polarized and anisotropic tensions from the neighboring tissues have a potentially big impact on morphogenetic processes. The anisotropic tension by the posterior midgut during gastrulation pulls on the lateral epidermis, which is visible by a corresponding AP stretching of the cells during the onset of germband extension . This anSimilar tissue interactions are essential for the morphogenesis of the amnioserosa and dorsal closure. The two sheets of the dorsal epidermis are exposed to an anisotropic tension from the pulsating and contracting amnioserosa as well as the contractile actin cable. Cell elongation occurs not only in the leading edge cells but also in the further distant second and third and so forth neighbors in the epidermis . It has DK wrote the manuscript and drew the figures. JG revised the manuscript. JG and DK conceived the study and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


No matter the age, social isolation is common among older adults due to a number of factors. In looking at social isolation through the framework of the International Classification of Functioning, Disability and Health (ICF) model, the biopsychosocial factors intersect. Care models will be explored that involve the ICF model multi-disciplinary concepts of body functions, activities, participation and environmental factors. Developing more programs to counter isolation is critical for the health of older persons. (1) The experience of vulnerability may be overcome by banding together through assisting others or building a peer support network. (2) A holistic perspective is needed in promoting interventions that support functionality. Regular programming with body awareness and cognitive reflection is enjoyed by institutionalized older adults. (3) The role of social action and social justice in reducing social isolation is part of training social work students about the importance of culture and advocacy. (4) Collected ethnographic data found that the practice of remembrance reduces social isolation regardless of the program. Gardening and storytelling were found to be opportunities to reduce social isolation. (5) With unexpected longevity in individuals with hemophilia due to scientific advances, researchers also found shame, fear, and coping through social isolation to avoid social assumptions of health status. Care models are being explored to support this cohort. To conclude, Dr. Sharon Bowland will summarize our abstracts and discuss how they revolve around the ICF model of care that can be applied to the important social determinant of health area of social isolation.


The aim of the study was to investigate the effects of neodymium: yttrium aluminium garnet (Nd:YAG) (1064 nm) and erbium: yttrium aluminium garnet (Er:YAG) (2940 nm) laser energy on titanium when delivered with conventional optics  or with a conical tip. Machined and micro-roughened implant discs were subjected to laser irradiation under a variety of energy settings either dry (without water) or wet (with water). Samples were scanned using a 3D non-contact laser profilometer and analysed for surface roughness, volume of peaks and the maximum diameter of the ablated area. Conical tip designs when used with both lasers showed no surface effect at any power setting on both machined and micro-roughened implant surfaces, regardless of the irrigation condition. When used with conventional delivery systems, laser effects on titanium were dose related, and were more profound with the Nd:YAG than with the Er:YAG laser. High laser pulse energies caused surface fusion which reduced the roughness of micro-roughened titanium surfaces. Likewise, repeated pulses and higher power densities also caused greater surface modifications. The presence of water reduced the influence of laser irradiation on titanium. It may be concluded that conical fibres can reduce unwanted surface modification, and this may be relevant to clinical protocols for debridement or disinfection of titanium dental implants. Titanium and its alloys are used commonly for dental implants. After insertion of a titanium dental implant, the accumulation of a dental plaque biofilm and an adverse host inflammatory response to this biofilm can lead to peri-implantitis, a condition where the bone around the implant is resorbed, and where if untreated the implant may be lost. Conventional treatments for peri-implantitis have included  mechanic2) laser [Lasers have been increasingly utilised in dentistry for the treatment both of periodontitis and peri-implantitis ,9,10. Th2) laser ,16 and v2) laser ,18 have 2) laser ,19.In the treatment of peri-implantitis, the erbium family of middle infrared lasers  has received the most attention. The laser energy from both is absorbed strongly by water . The Er:Several studies have reported no significant change in the surface roughness of implants irradiated with Er:YAG lasers at intensities from 60 mJ/pulse to 180 mJ/pulse (average powers less than 1 Watt) and application times of up to 2 min ,25,26. PThe effect of any given laser depends not only on the absorption of the laser energy into the substrate but also on delivery system used, which changes the power density and may in some cases also change the pulse shape . With miOther factors that may influence the effect of laser energy on a substrate include the water-layer thickness if a cooling water mist spray is used , the pulThe primary aim of this study was to assess the effect of conical fibre tips versus conventional laser delivery systems (plain fibres and focusing handpieces) on the nature of the interaction of infrared laser energy with titanium. A secondary aim was to assess the influence of water irrigation on the interaction of laser energy with titanium. The primary laser of interest was the Er:YAG laser (2940 nm). An Nd:YAG laser (1064 nm) was included in the study as a positive control, since it is known to interact strongly with titanium and many other metals. It was hypothesised that the extent of surface modification of titanium surfaces would be reduced by the use of conical fibre tips and by the presence of water.Two free running pulsed laser systems were used: Nd:YAG  and Er:YAG . The Nd:YAG laser was fitted with a 200 μm plain glass fibre  which was used either with a plain (cleaved) end, or after undergoing a conical honeycomb modification through a process of etching and abrasion as described previously  to incre® with plus surface, Dentsply Friadent GmbH, Steinzeugstr, Mannheim, Germany). Discs were cleaned with ethanol prior to the study to remove traces of machining oil or debris.Discs of grade IV commercially pure titanium (Ti), as used in dental implants, were used . These discs were 10 mm in diameter. The discs had a plain machined surface or had been micro-roughened by the manufacturer using grit-blasting and thermal etching to impart the desired microstructure for dental implants  for the Er:YAG laser-focusing handpiece. Multiple laser treatments were undertaken on non-exposed regions of each disc.To explore the effect of a surface film of water, discs were irradiated dry (without water) or wet (with water). For the latter, a micro-pipette was used to transfer a 10 μL droplet of de-ionised water onto the surface of the discs immediately before laser exposure. The study followed a matrix design, and involved two different laser types (Er:YAG or Nd:YAG), two different fibre tip designs , and two different irrigation conditions (wet or dry). With the Er:YAG laser, a focusing handpiece was available, so this was also included as a further experimental group. Untreated discs and untreated areas of discs served as controls. The experimental groups are summarized in 2) was recorded over three seconds and the average value was used . The power density  using a 20× lens. Profilometric analysis was completed only for samples where laser irradiation produced a visible zone of ablation. Surface roughness was measured for all samples. This parameter quantifies the Z-axis perpendicular to the analysed surface (according to ISO 25178) . The proThe surface roughness (Sa) of each sample before and following irradiation was compared using paired sample t-tests, for each of the gradations in laser parameters. Effects of irrigation on surface roughness, ablation zone volume and maximum diameter were compared for each laser parameter using a similar approach. Within each treatment group, the effects of laser parameters surface roughness, crater volume and maximum crater diameter were assessed for normality using the Kolmogorov–Smirnoff test and then analyzed by using ANOVA, with Bonferroni post-hoc tests. The level of significant was set at α = 0.05.p = 0.0002).Prior to any laser treatments, the surface roughness of machined (smooth) surfaces  was significantly less than that of roughened surfaces , as supplied by the manufacturer (Changes in mean surface roughness for machined (smooth surface) and rough surface samples irradiated with the Er:YAG laser using a focusing handpiece are shown in Conical fibre tip designs when used with the Er:YAG laser showed no surface effect at any laser parameter setting on both machined and micro-roughened implant surfaces, regardless of the irrigation condition. These groups are therefore not discussed further.As shown in The reduction in surface roughness for rough samples b was lesConical tip designs, when used with the Nd:YAG laser showed no surface effect at any laser parameter setting on either machined or micro-roughened implant surfaces, regardless of the irrigation condition. These groups are therefore not discussed further.Exposure to the Nd:YAG laser increased the surface roughness of smooth Ti discs under wet and dry conditions, and this effect was variably attenuated by a film of water a. On rouThe total volume of peaks within the ablation zone was measured for samples treated at 300 mJ and above, as these gave a measurable zone of ablation. For smooth discs, there was no significant relationship between pulse energy and the volume of peaks, and this outcome variable was not influenced by the presence of water. For rough surface discs, there was no significant relationship between pulse energy and the volume of peaks for wet conditions, but there was a significant positive relationship for dry conditions. Water reduced the peak volume only at 300 mJ. Measurements of volume or of the diameter of the ablation zone were not possible as no visible zone of ablation was produced on the titanium surface. On both machined surfaces and roughened surfaces, there was a significant positive relationship between pulse energy and the volume of peaks for both wet and dry conditions. The presence of water had a variable influence on smooth samples, with a trend to decrease the volume of peaks for rough surfaces at higher pulse energy settings.For smooth surfaces, under wet conditions, power level did not influence the maximum diameter of the ablation zone. Under dry conditions the diameter was significantly decreased at 400–600 mJ. For rough samples, 500 mJ and 600 mJ power resulted in significantly greater diameter of the ablation zone. Measurements of diameter of the ablation zone for Group 2 was not possible as no visible zone of ablation was produced on the titanium surface.The diameter of the ablation craters increased with pulse energy, on both smooth and rough surfaces. The presence of water reduced the diameter of the zone of ablation for both surface types. This study provides several insights into the interactions between near infrared and middle infrared laser energy and titanium, with the former being strongly absorbed, and the latter only weakly absorbed. The titanium discs provided by the manufacturer are consistent with the surfaces provided on implants for clinical use, with the smooth surface being typical for abutment components, and the roughened surface being representative of the surface of the implant fixture below the alveolar bone crest. Indeed, the roughened discs displayed almost twelve times the roughness of machined discs. The purpose of this implant surface modification is to increase the surface area available for apposition of bone during osseointegration of the implant ,41. The The Nd:YAG laser was used as a positive control in this study. While this laser induced surface modifications to both types of titanium surface when the plain optical fibre tip was used, no modifications occurred when the modified conical honeycomb tip was used. Based on this, the original hypothesis that the use of the conical tip would reduce the adverse effects of laser energy on the titanium surface is supported.Despite previous research indicating the deleterious effects of Nd:YAG lasers on implant surfaces ,43,44, tWith regard to the Er:YAG laser, its effects on titanium surfaces were less than those of the Nd:YAG laser. Overall, the current findings are consistent with previous research demonstrating no significant surface effects following irradiation with the Er:YAG laser at up to 140 mJ/pulse at 10 Hz on roughened (SLA surface) implants  and on mWith regard to the role of water, under clinical conditions, typical pulse energies are in the order of 50–200 mJ, and the Er:YAG laser is used with a water mist spray. The Er:YAG laser, when used at low pulse energy with a water mist spray, was found to effectively remove experimentally induced biofilms from SLA implant surfaces at 80–100 mJ/pulse (10 Hz), without significant alteration to the surface ,46. In tThe results of the present study also show the effect of laser spot size and thus power density on Er:YAG laser effects on titanium, with the focusing handpiece causing significant effects for the same pulse energy, compared to the plain optical fibre. Plain fibre ends give a beam dispersion of around 20°. Moreover, the gallium doped endodontic fibre used in the current investigation attenuates the laser energy within the fibre, to a greater extent than occurs with the sapphire window of the focusing handpiece . Any attAn important variable that needs to be taken into consideration is the spot profile. The present laser had a Gaussian beam profile. Some dental Er:YAG lasers have complex beam profiles because of multimode operation, and this spatial inhomogeneity may influence the geometry of surface craters on the substrate ,48. The A key point of interest in the current study was the lack of appreciable surface modification or deleterious effects from either laser when modified conical fibre tips were used. This can be explained by the reduced beam intensity in a frontal  direction (by as much as 49%) and correspondingly greater lateral emission of laser energy ,49,50. TThe generalization of the current findings to the clinical setting should be performed with caution. The 90-degree angulation of the laser beam to the titanium surface used in the study would not necessarily be the same as for clinical practice. The results would also vary for other brands of lasers, because of differences in pulse shape and pulse duration. Hence, the current results may not be directly applied to other brands of Nd:YAG and Er:YAG laser systems on the market. Further, the impact of crevicular fluid, blood and saliva may not be the same as for water. The presence of plaque biofilm and calculus on laser interactions with titanium was outside the scope of this laboratory study.Pulses of laser energy from Er:YAG and Nd:YAG lasers may modify the surface characteristics of machined and roughened dental implants, depending on the pulse energy and the power density. The Er:YAG laser delivered using a plain fibre tip was associated with minimal surface modification, while the Nd:YAG laser used at similar parameters caused considerable changes. In contrast, when a modified conical tip was used with either laser type, no surface melting or cratering occurred on either machined or micro-roughened titanium surfaces. Using such tips for implant debridement or disinfection may help prevent undesirable surface modification.