The metal-free cousins of metal-organic frameworks, covalent organic frameworks (COFs), are a class of pre-designable crystalline polymers composed of light elements and connected by strong covalent bonds. COFs are being given more and more attention in the electrochemical sensor field due to their fascinating properties, such as highly tunable porosity, intrinsic chemical and thermal stability, structural diversity, large specific surface area, and unique adsorption characteristics. However, there are still some key issues regarding COFs that need to be urgently resolved before they can be effectively applied in electrochemical sensing. In this review, we summarized recent achievements in developing novel electrochemical sensors based on COFs, and discussed the key fundamental and challenging issues that need to be addressed, including the mechanisms underlying charge transport, methods to improve electrical conductivity, immobilization methods on different substrates, synthesis strategies for nanoscale COFs, and the application of COFs in different fields. Finally, the challenges and outlooks in this promising field are tentatively proposed. Covalent organic frameworks (COFs), a new class of multifunctional porous crystalline materials, are two- or three-dimensional (2D or 3D) porous crystalline materials built by light elements  via strong covalent bonds   and a counter electrode separated by a thin electrolytic layer  transport  Xie et . In hoppThe low intrinsic conductivity of COFs still imposes a great challenge for their applications in electrochemical sensing  were synthesized on an amino-functionalized carbon nanotube (NH2-f-MWCNT) from TTA and DHTA via imine linkages , which can be synthesized through either direct synthesis or post-synthetic metalation by using planar and large π-electronic macrocycles as the building and paring units for the metal  porphyrin)-COFs (POR-COFs) with a high crystalline order were electrochemically synthesized via the formation of phenazine linkages by controlling the temperature, potential scanning rate, and electrode materials and co-crystallization with pyridine  with the aid of acetic acid and polyvinylpyrrolidone (PVP). Unlike traditional solvothermal COFs synthesis, this approach does not need any vigorous reaction conditions, such as a solvothermal and inert atmosphere. More importantly, scaling up to a gram-scale nanoscale COF synthesis was easily achieved. In order to decrease the size of COFs, a template-mediated synthesis of hollow tubular COFs using a twostep strategy was reported by Pachfule et al.  with a different surface morphologic structure is reported by facile ambient temperature synthesis, which shows the advantages of higher surface area, good water dispersity, long-term stability, excellent electrical conductivity, and pre-concentration effect  of 0.2361 μM and 0.6568 μM, respectively.Endowed with an intrinsic absorption capability, COF-based electrochemical sensors have been widely used in the detection of environmental pollutants including hydrazine, explosives, catechol, nitrophenol, hydroquinone, bisphenol A, paraquat, and heavy metals. Porous and redox-active COFs were demonstrated to remove and detect hydrazine  were synthesized, which are a potential candidate for the sensitive detection of target cancer markers or living cells (EGFR and living Michigan cancer foundation-7)  benzene (TFPB) and thionine (Thi) and this porous crystalline material was a highly ordered 2D nanosheet. A highly selective and sensitive electrochemical sensing platform based on 2HP6@Au@CP6@COFs was successfully established for the determination of dangerous and explosive sodium picrate (SP)  support at room temperature via π-π interactions. The COFs-CNT composites modified electrode showed a high specific surface area (147.3 m2 g−1), and excellent electrical conductivity, which exhibited an excellent analytical performance for the detection of the nitrofuran antibacterial agent furazolidone  in their frameworks, or hybridizing COFs with other electroactive components with following scheme in p−FeporNH2−BTA) was synthesized via aldehyde-ammonia condensation reaction between 1,3,5-benzenetricarboxaldehyde and 5,10,15,20-tetrakis(4-aminophenyl)-21H, 23H-porphine, followed by post-modification with Fe2+. The synthesized COFp-Fepor NH2-BTA showed a good electrochemical redox property and electrocatalytic activity toward the reduction of hydrogen peroxide  for the detection of hydrogen peroxide. Recently electroactive COFs were used to construct a ratiometric electrochemical sensor. Wang reported an electroactive COF with multiple redox-active states synthesized by an amine-aldehyde condensation reaction between 4, 4′,4″- trianiline and 2,5-dihydroxy terethaldehyde (COFDHTA−TTA), which was applied for the construction of a ratiometric electrochemical sensor for the detection of hydrogen peroxide and pH level based on both current and potential signals  novel synthesis strategies  need to be developed for conductive, nanoscale, and electroactive COFs for enhanced sensitivity and electrocatalytic activity. (2) Finding a simple and efficient surface modification method/strategy on conductive substrates is an urgent problem that needs to be resolved in the future. (3) The intrinsic fragility, powdered crystalline state, and large size of COFs lead to a low active area, low mass transfer rate, and difficult modification as well as poor stability on the electrode. Therefore, simple and facile synthesis methods for nanoscale and hydrogels/aerogels COFs need to be further investigated. (4) The antifouling capability and biocompatibility of COF-based materials still need to be studied and improved for the analysis of biological samples. (5) With the enhanced thermal, chemical, and mechanical stability, biomolecules encapsulated in COFs may be further explored to broaden their operational conditions and extend their potential applications in electrochemical sensors. (6) Molecularly imprinted COFs used in electrochemical sensors need to improve in their selectivity and sensitivity. (7) It will be an efficient method to improve the performances of conduction and sensing by using COFs as nanocarriers to encapsulate organic molecules that can be released through specific stimuli (Chang et al., SC writing-original draft, table preparation, copyright application, and manuscript figure. BY writing-original draft, manuscript concept and design, funding acquisition, manuscript revision/review/editing, and manuscript figure. GL and DZ review/editing/revision. 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.


Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as (1) they are a single-component system i.e., their light sensing and ion-conducting functions are encoded by the 7-transmembrane domains and, (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator, and the effector components are involved in attaining synchronous regulation of signaling. Optical modulation of the multicomponent network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanylate cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish a proof of concept for utilization of complex rhodopsin (modular rhodopsin) for optogenetic applications. Light sensor coupled modular system could be expressed in any cell type and hence holds great potential in the advancement of optogenetics 2.0 which would enable manipulating the entire relevant cell signaling system. Here, we had identified 50 novel modular rhodopsins with variant domains and their diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized based on their sequence and structural homology with previously reported rhodopsins. The presented novel modular rhodopsins with various effector domains leverage the potential to expand the optogenetic tool kit to regulate various cellular signaling pathways across the diverse biological model systems. The photobehavioural responses of many organisms are mediated by the rhodopsin-based photoreceptor(s) that are distributed across almost all clades of life. Rhodopsins are seven-transmembrane helical proteins that use retinal as a chromophore. Based on the isoforms of the retinal bound in the ground state, rhodopsins are classified into two broad categories i.e., Type I or microbial type (MTR) and Type II or animal-type rhodopsins (ATR). MTRs are widely distributed across all kingdoms of life and perform diverse physiological functions, such as the light-activated ion pump Bacteriorhodopsin (BR)  and HaloATR or type II rhodopsins are broadly classified as vertebrate and invertebrate rhodopsins based on variation in their amino acid sequences . The ATRSalpingoeca rosetta in Xenopus oocyte and HEK293 cell lines demonstrated the light-activated cGMP and cAMP-phosphodiesterase activity [Blastocladiella emersonii and Catenaria anguillulae when expressed in various mammalian cell lines, could generate substantial cGMPs [Enzyme-rhodopsins  have emerged as promising optogenetic tools for the precise and non-invasive spatiotemporal control of cyclic nucleotide signaling pathways. The heterologous expression of RhoPDE ,10 from activity . Similaractivity ,13 isolaal cGMPs ,15. Furtal cGMPs . Consequal cGMPs ,17,18 asChlamydomonas rhodopsin as a template. The rhodopsin identity, sequence accession number, homology, conserved domains, are summarized in http://www.mbio.ncsu.edu/bioedit/bioedit.html). All color editing was done by using the BioEdit program. The rhodopsin domains of new MTRs were identified by sequence alignment with canonical rhodopsins, analyzed with conserved domain architecture retrieval tool (CDART) [Extensive genome database search for MTRs and modular rhodopsins were performed on the JGI genome database, metagenome database, and NCBI portal using BR and  (CDART)  and cons (CDART)  programsMolecular evolutionary analysis of typical MTR and rhodopsin domains (helices 1−7) of modular proteins was performed computationally with protein sequences. Multiple sequence alignment of the rhodopsin domain was done on Clustal X 2.0 . PhylogeThe interactomes of effector domain(s) associated with ChRs, i.e., FimV, MED15, and UL36, were constructed. The interacting partners for each of the effector domains were predicted using the String version 11  and the C. reinhardtii is an excellent system to study and learn various aspects of cell biology ranging from the photobehavioural responses  to photosynthesis, cilia biology, intraflagellar transport to vesicle, and membrane-bound trafficking and dynamics [Chlamydomonas led to the early discovery of modular rhodopsins in this green alga but since then very few have been reported in other organisms.Mining the genome database of the organisms from diverse taxa and strata revealed the presence of MTRs in various organisms inhabiting diverse habitats from freshwater to terrestrial environments. The phototactic green alga dynamics ,27. SteaHere, we have identified new microbial modular ChRs A,B and mKlebsormidium nitens, TsRh1 from Tetraselmis subcordiformis, and GpRh1 from Gonium pectorale. KnRh3 is coupled with the peptidoglycan binding protein, FimV, whereas TsRh1 is the blue-shifted ChR for which the rhodopsin domain has been characterized [Gonium pectorale is coupled with UL36 (large tegument protein). The optogenetic potentials of these modular domains  are summarized in Our targeted search for the modular ChR yielded three modular ChRs as shown in cterized . TsRh1 icterized , howeverAll the three ChRs have the conserved seven transmembrane domains and the lysine motif at the seventh helix that forms a covalent linkage with retinal B. Asp253The conservation of important amino acids reflects their functionality and could be engineered to enhance their properties. Thus, newly identified modular ChRs hold the potential to be used as optogenetic tools for controlling new biological pathways. C. reinhardtii [Apart from the three modular ChRs, the genome database search also led to the identification of many modular sensory-type rhodopsins from different alga. A diverse set of domains fused with modular sensory-type rhodopsins were identified in a single ORF, which suggests multiple light-mediated cellular signaling pathways in these algae. Most of the identified rhodopsins are coupled with the two-component histidine kinase (HisK) and response regulator (RR) system. The first modular rhodopsin identified and characterized was Chlamyopsin5 (Cop5/HKR1) of nhardtii .C. reinhardtii, with dichromic absorbance maxima in the UV range however, their native functional role is still not clear [C. reinhardtii and other algae as well. Cop6–8 expressions were further confirmed in C. reinhardtii and Cop8 was localized in cilia and eyespot in a light-dependent manner [Volvox carteri and other algae . The amino acid residue at the 105th position of the highly homologous green absorbing proteorhodopsin (GPR: AY210898) and blue absorbing proteorhodopsin (BPR: AY210919) have nonpolar leucine and polar glutamine residues, respectively. The substitution of either convert it into the other form and vice versa . The fouIn addition to blue and green-shifted MTRs, the red-shifted MTRs ,42,43,44MTRs provide a smart alternative pathway of ATP production, other than photosynthesis, in archaea and help in the survival of the organism in harsh conditions. Many reports have been published regarding the evolutionary pattern of MTRs ,49 but tChlorella subellipsoedea. Sequence alignment also confirmed the presence of important residues required for pump activity in AsRh4  were grouped with ChR and VChR  while rhe of ATP . The phoironment . OtHKR/Oironment . The cha2+ or Mn2+. The conserved motifs, especially the transition state stabilizing residues of the cyclase are also missing, which suggests that other transition state stabilizing molecules might be involved in signaling  serves as a signaling molecule in many prokaryotes and eukaryotes. Based on the substrate specificity, there are two classes of cyclases—adenylyl cyclase (AC) and guanylyl cyclase (GC). Multidomain cyclases are generally composed of a receptor domain at the N-terminus and a cyclase domain at the C-terminus with a kinase homology domain in the center. A similar architecture is found in modular rhodopsin coupled cyclases. Sequence analysis suggests that most cyclase domains have a conserved amino acid residue to perform the enzymatic activity. Cop5 and Vop5 lack the conserved aspartate involved in metal binding . Substraignaling . Both moC. reinhardtii, cAMP induces the rapid mobilization of membrane adhesion receptor protein from the cell membrane to the ciliary membrane in gametes [C. reinhardtii, cyclase activity biases the photo-behavioral response and carotenoid biosynthesis [Ostreococcus lucimarinus, symbiotic algae, and in colonial algae Volvox carteri. The rhodopsin coupled guanylyl cyclase from the fungus Blastocladiella emersonii was required for the phototactic behavior of the zoospore and had shown in vitro functional activity as well [Xenopus oocytes, hippocampal neural cells, and Chinese hamster ovary cells [In  gametes  which le gametes . In the ynthesis . The modynthesis . These a as well . Rho-GC ry cells ,13. It wPseudomonas aeruginosa, FimV is an inner membrane hub protein that controls the type IV pilus (T4P)-mediated twitching motility by regulating the intracellular cAMP level via activation of the adenylate cyclase CyaB [P. aeruginosa, where PilG may regulate the directional movement, while FimV functions to localize both structural and regulatory elements to the cell poles for an optimal function [Among a variety of effector domains coupled with the ChRs, we selected the FimV, MED15, and UL36 domains of functional importance, which have not yet been characterized in the algal system. We subjected these domains to protein-protein interaction network analysis and identified their potential partners and associated pathways. The protein-protein interaction analysis for the FimV domain revealed its association in regulating bacterial pathogenesis machinery . In the ase CyaB ,54. Factase CyaB . HoweverCaenorhabditis elegans orthologue, MDT15, and yeast orthologue GAL11p [MED15 (co-activator) plays a crucial role in the transcriptional regulation of RNA polymerase II-dependent genes . The proe GAL11p ,58. It he GAL11p ,59. Sevee GAL11p ,59. In be GAL11p . The locG. pectorale is a tegument viral protein found in herpes simplex virus 1 (HSV-1) and its homologs are well distributed across the members of Herpes viridae [The UL36 domain, associated with modular ChR, GpRh1 from  viridae . UL36 pr viridae  which is viridae ,64,65. P viridae . In huma viridae ,68. UL36 viridae . Interes viridae . In the In this study, we have reported various rhodopsins with diverse effector domains. Based on multiple protein sequence alignments and phylogenetic analysis, these modular rhodopsins can be categorized as ChRs, ion pumping (AsRh4), or sensory-type rhodopsins. Owing to the diverse functions offered by the encoded effector domains of these modular rhodopsins hold great potential to expand the optogenetic toolkit. We have also proposed the working models of the modular channelrhodopsins , in regulating processes ranging from bacterial pathogenesis, transcription to viral replication and light-gated proteasomal regulation, respectively. The established methods for expression and delivery system could be systematically utilized to design further experiments to study the modular rhodopsin mediated optogenetic modulation of crucial processes across the biological systems. These naturally occurring light-sensitive rhodopsin modules could be recruited in the biological systems and activated relevant approaches such as forced conformational change, heterodimerization, etc. These conformational changes could bring desired changes in cellular signaling like gene expression, protein translocation, and receptor signaling pathways. Empirical optimization, targeted engineering, and directed evolution of the modular rhodopsin(s) would enable us to refine light-sensing mechanisms  and engineering of the coupled effector domain(s) for extensive applications avenues in optogenetics.


On the basis of our results including the shapes, locations and volume changes of the large collapse, evidently a new vital piece of information was obtained so that it could be used to interprete the sixth nuclear test more accurately.The artificial earthquake of mb 6.1 related to the North Korea’s sixth nuclear test occured at Mt. Mantap, North Korea on September 3, 2017. It was reported that a large and complex surface deformation was caused by the event. The surface deformation was composed of expansion of explosions, collapse, compaction and landslides. Since the precise vertical deformation measurement is very important to estimate the stability of the nuclear test facility, we retrieved a precise 3D surface deformation field and then decomposed the vertical deformation pattern from the 3D deformation. The measured maximum deformation was about − 491, − 343 and 166 cm with the measurement uncertainty of about 3.3, 4.1 and 2.7 cm in the east, north and up directions, respectively. The maximum horizontal deformation was approximately 515 cm. The horizontal deformation clearly showed a radial pattern because it was mainly caused by the explosions and landslides, while the vertical deformation displayed a rugged pattern because it was affected by the explosions, compaction and collapse. The collapse may seem to occur along the underground tunnels and at the test site’s epicenter as well. Moreover, the severe collapse was observed westside from the epicenter of the sixth nuclear test, and it has a depth of about 68.6 cm on the area of 0.3765 km After 8 min 32 s from the sixth test, the second earthquake of smaller magnitude occured2. Many research groups including USGS have considered the source of second earthquake as a large collapse, which also raised up questions on the stability of the infrastructures of the test site under the Mt. Mantap. The infrastructures in seismically active area usually experience irrecoverable damages and ground subsidence6. Measuring surface subsidence could be used to estimate the ground stablility7. However, there have been no valid measurements about the collapse or ground subsidence in the North Korea Nuclear test site because of a paucity of information about the North Korea nuclear test9.North Korea has conducted six nuclear tests at Mt. Mantap, Pungyeri, North Korea since 2006. The seismic magnitudes estimated from the North Korean’s nuclear tests has been increased since it’s first test. The magnitude of the sixth test was mb 6.1, which is the biggest one among the six tests. Due to North Korea’s sixth underground nuclear tests, large and complex deformations occurred, which also caused landslides around the test site13. A lot of geological events have been measured by using SAR Interferometry (InSAR) or offset tracking (OT) methods such as volcano16, earthquakes18, landslides20, land subsidence22 and glaciers26. Among them, land subsidence is especially successful and traditional application of SAR deformation measurement31. For example, risk assesments of underground voids were performed via multiple-temporal InSAR. Falorni et al. introuduced surface deformation rate variation according to mining and rail tunnel excavation progression30. Rabus et al. monitored land subsidence on the subway and water pump tunnels31. Since InSAR was firstly applied to Nevada nuclear test site to measure the surface deformation, several studies have been reported about observing the nuclear test sites using InSAR34. Wang et al. (2018) observed the deformation from the 2017 North Korea nuclear test using SAR measurements and suggested a reasonable deformation scenario1. However, there have been no deformation analysis about large surface collapses induced by the 2017 North Korea Nuclear test. In order to measure large collapses in North Korea’s test site, three main issues need to be considered.Synthetic aperture radar (SAR) is a unique method that can generate deformation map with milimeter to centimeter precision37. The biggest explosion inevitably caused complex and steep surface deformation which exceeds the phase difference of 2 38. Thus, InSAR could not be utilized to describe the deformation nearby the test site34. The OT method is an alternative aprpoach to measure the large and complex surface deformation induced by the 2017 North Korea Nuclear test because the method is allowed to measure the complex and steep surface deformation by using intensity cross-correlation approach38.First, InSAR measurements near the test site was too decorrelated to map the deformation1. Therefore, the spatial resolution and precision of the measurements are very important to recognize the spatial distribution of the ground subsidence. The precision and resolution of the OT measurements from a single search kernel largely depends on the search kernel size39. When the window size is small, the detailed correlation peak could be estimated well, therefore the resolution will be higher. However, in case the search kernel size is too small, the measurement precision will be much lower. On the other hand, if the kernel size is larger, the number of effective measurements must increase, but the resolution will be much lower. The trade-off relation needs to be overcome. In case of Wang et al. (2018), they generated OT measurements with large kernel to enhance measurement precision, since they focused on estimating explosive source using SAR data. Thus, their measurements were too smoothed to recognize the surface collapses1.Second, the subsidence from the second event was known as occupying only localized area of the deformation fields41. The geometry of the OT measurements from the one path SAR represents two-dimensional (2D) deformations in the range and azimuth directions, which is respectively line-of-sight (LOS) and along-track (AT) deformations. The LOS measurement includes both the horizontal and vertical components of surface deformation42. Thus, the vertical deformation should be decomposed from the LOS deformation, because the horizontal and vertical deformations were complexly mixed in the LOS deformation1.Third, three-dimensional (3D) decomposition were essential to measure the localized land subsidence38. The two ALOS PALSAR-2 stripmap interferometric pairs were acquired from the ascending (20170829_20170912) and descending (20170831_20170928) orbits, and the pairs were utilized to measure the ascending and descending LOS and AT deformations. Then 3D deformation field was retrieved from the ascending and descending LOS and AT deformations. Finally, the spatial patterns of the horizontal and vertical deformations were separately analyzed.In this study, we show the evidence of large collapse induced by the 2017 North Korea Nuclear test. For that, a multi-kernel OT (MKOT) approach was employed to overcome the limitation of the trade-off between measurement precision and spatial resolution44. The underground tunnel network from the north portal is connected to five other test sites45. It means the long and complex underground cavity had been constructed. If the ground stability of the tunnel is poor, it is possible to recognize the deformation pattern of a linear form46. Figure 2. Especially the vegetation on the top and ridge of Mt. Mantap disappeared, as one can see in Fig. Figure 47. The incidence angles of the ascending and descending pairs were about 42.9 and 36.2 degrees, respectively. We can expect that the descending pair had a more severe geometric distortion (i.e. foreshortening effect) due to the relation between the incidence angle and topography 47.Table The two co-seismic interferometric pairs were processed by (1) the azimuth common band filtering and sub-pixel-level registration, (2) multiple offset estimation, and (3) initial azimuth and range OT generation using the 3D median filtering. The offset was calculated every 4 pixels in the azimuth and range directions to reduce the computation time. The step of 4 pixels in the offset estimation corresponds to the 4X4 multi-looking in the InSAR method. Thus, the pixel spacing in the measured OT maps was about 7.4 × 8.4 m in azimuth and range directions, respectively. The kernel sizes were designed from 96 × 96 to 256 × 256 pixels with the interval of 32 pixels in both the azimuth and range directions, respectively. Thus, a total of 36 OT measurements were calculated by using the kernel sizes of 96 × 96, 96 × 128, 128 × 96, 96 × 160, 160 × 96, 128 × 160, 160 × 128, …, 224 × 256, 256 × 224.38.Figure 47. The patterns must be reduced by an ionospheric correction method. And, the ascending range OT maps were almost flat except the deformation area  as seen in Fig. 47. Figure 48. Ionospheric effect shows azimuth streaking on the SAR-derived deformation maps in general. Applying large-kernel directional median filter along streaking direction can mitigate high-frequency signal  and generate ionospheric phase screen48. To estimate the ionospheric component, the steep deformation area on Mt. Mantap was first masked out, and then the directional median filters were designed for the ascending and descending azimuth OT maps, respectively 49. The maximal measured deformation was about − 266.9, − 318.3, 482.6, and 328.9 cm in the ascending azimuth and range OT maps and the descending azimuth and range OT maps, respectively  because the estimated 3D maps have a higher resolution and precision via the MKOT approach25.Figure 8. However, the validation is an important process to enhance the confidence of the measured 3D deformation field. In case of no confident validation data, the measurement performance has been estimated by calculating the mean and standard deviation of stable25. Thus, to validate the measurment performance of the measured 3D deformation, the mean and standard deviation of each component of the 3D deformation were calculated in the non-deformed area, which was selected as the outside of dotted black box on Fig. The in-situ measurements was almost never released, and hence the validation with a in-situ confident data on this area is realistically impossible49. The standard deviations were about 3.3, 4.1 and 2.7 cm in the east, north and up directions, respectively. The standard deviation in the north direction was slightly larger than others because the north component is largely dependent on the azimuth measurements (1) that were contaminated by ionospheric effects  and (2) that had a lower precision than the range measurements49. This histogram analysis indicates that the measurement performance of the retrieved 3D deformations is reliable with centimeter precision.Figure Figure Thus, the direction of the measured horizontal deformation was compared with the topographic aspect calculated from 1 arc-second shuttle radar topography mission (SRTM) DEM. Figure 50. The underground tunnel network had been constructed from the north portal to the nuclear test positions under Mt. Mantap51. Thus, we can assume that the relatively severe vertical deformation and spatially linear subsidence pattern were caused by underground artifical void space such as tunnel.Figure 1, the components were mixed in the measured deformation. Thus, if the magnitudes of collapses or compactions are locally smaller than that of expansion, the vertical deformation would show a positive value (uplifts) irrespective of downward movements. On the other hands, if the downward components are larger than the upward component, the vertical deformation would have a negative value (subsidence), but it can be underestimated due to the expansion component.Figure The collapse area was decided from the vertical deformation by calculating the deformation rate. Figure 1, and the location of tunnels (grey dotted lines) from 38 North45. The subsidence areas were divided into the A, B and C zones. In the zone C, there were a spatially linear pattern extending to west, north and south from the location of implosion1. Although the uplift signal were measured between the zones B and C, it is possible to have a connection between the zones B and C. Likewise, it has a high possibility that the zones A and C would be connected although the subsidence pattern was partially discontinued in the area of dotted black box on Fig. The subsidence areas were marked in Fig. 2, respectively. The areas of the zones A and B were similar, but the area of the zone C was about 6 times larger than both the zones A and B. And we also calculated the volume change on the zones A, B and C to approximately estimate the total amount of the collpase. Before the volume change calculation, the mean deformation of the each boundary was calculated to set a reference point of the deformation because the vertical deformation included complex up and down deformation components49, and the volume change was calculated by compensating the reference vertical deformation. The volume changes in the zones A, B and C were about − 2.58E + 05, − 1.04E + 05 and − 4.28E + 05 m3, respectively. The volume changes in the zones A, B and C are same amount of homogeniously subsiding 36.2, 14.5 and 60.0 m in the area of a soccer field, respectively. Despite the area difference of A, B and C, the volume change of the zone C was about 1.7 and 4.1 times greater than the zones A and B. Moreover, the mean subsidences from the reference vertical deformations were about − 68.6, − 32.9 and − 20.9 cm in the zone A, B and C, respectively. From the results, it can be easily recognized that most severe subsidence occurred in the zone A. It should be noted that the zone A must be considered as a severe collapse, or if not, at least be monitored to check long-term geological stability46.The zones A, B and C have areas of about 0.3765, 0.3146, and 2.0477 km51. In other words, the branching patterns of the tunnels were similar, but the locations were not exactly the same51. The discrepancy is still controversial, and hence additional analysis needs to be performend by using various measurements or documentations. Despite these discrepancies, we believe that our surface displacement measurement could suggest a possible collapse of the tunnel under Mt. Mantap .The location of the tunnel and tunnel-related subsidence area show a discrepancy. This is because we manually situated the locations of the tunnel and test sites using the photo from 38 North because we didn’t have documented information about the cordinates of the underground structuresRadar data have been successfully used to monitor surface deformation because they have made it possible to measure the surface deformation changes with submeter accuracy. The phase information of the radar data enables the precise measurement. However, the phase information has an inevitable disadvantage that it cannot be applied to large and complex surface deformation areas since the measurement values of adjacent pixels must be present at half the radar wavelength. Normally, the radar wavelengths are about 3, 6 and 24 cm in X-, C- and L-band radar data respectively. If deformation difference among adjacent pixels exceeds half of the radar wavelength, no valid radar phase information can be obtained. In this case, instead, the intensity information of the radar data has been widely used. Conventional methods have low precision over 10 cm and low spatial resolution over 100 m, but they are nevertheless employed to estimate the surface deformation changes from the intensity as those methods can measure the large and complex surface deformation.1. Thus, the decomposition of the deformation is the most crucial step in esimating the explosive yield from the nuclear detonation. If the step of decomposition is ignored, the explosive yield is most likely to be either overestimated or underestimated. In spite of that, the estimated explosive yield of 191 kilotonnes of TNT equivalent and the source depth of 450 m has been already reported without the necessary deformation decomposition1. It is fairly reasonable to expect the estimated explosive yield to be either overvalued or undervalued in that the surface deformation measured in the previous studies had low accuracy and spatial resolution. Unavoidably, the measured deformation only showed an overall deformation pattern in the absence of a decomposition process.The surface deformation related to North Korea’s sixth nuclear test was very large and complex, and it consisted in explosions, collapse, compaction and landslidesAs discussed above, one of the most essential things in monitoring nuclear tests is to recognize the detailed local deformation pattern. The detailed local deformation pattern permits not only analyzing the spatial distribution of the deformation components but also decomposing the deformation components. In this study, the precise local deformation was measured by the MKOT method. The MKOT measurements have been well known to deliever distinctly improved performance such as several centimeters of accuracy and tens of meters of spatial resolution. When 3D deformation field was retrieved from the MKOT measurements, we could find (1) that the horzontal deformation was mainly mixed by the deformation components from explosions and landslides and (2) that the vertical deformation mostly included the deformation components of explosions and collapses.The horizontal deformation vector showed the explosion-induced isotropic deformation pattern. The horizontal deformation became larger closer to the epicenter of the sixth nuclear test, and the deformation concentrically decreased as the deformation areas moved far away from the epicenter. However, since the epicenter is adjacent to the top of Mt. Mantap, the topographic slope vector related to landslides showed a horizontal deformation pattern similar to an explosion-induced deformation pattern. Unfortunately, separation of horizontal components between the landslide-induced and explosion-induced deformation vectors is not easy because of similarities in their deformation patterns. The nuclear test facility might have been designed the way it was, taking these deformation similarities into consideration. Anyway, it is true that the deformation similarity may lead to overestimation or underestimation of the nuclear explosive yield.1. The maximum collapse came to over 100 cm, and it would even amount to 250 cm if the extended uplift of 150 cm was counted. The large collapse can be caused by either natural underground caves or artificial underground facilities. However, the collapse under consideration were way too big to say that they resulted from natural underground caves. This collapse also has a linear subsidence pattern as shown in Fig. The vertial deformation did not show the isotropic deformation induced by the explosion because the collapse areas were very large45. In our results, the largest collapse was found in the collapse area A in Fig. The second earthquake after a main shock would be very likely caused by this collapse. However, no such underground facilites in the area A were identified from the large scale topographic map that the North Koreans displayed1. Those serial process would attenuate the explosion-directly-related deformation components. In other words, the measured vertical surface deformation is underestimated to estimate explosive yield. In spite of that, the explosive yield has been estimated without excluding the vertical deformation1. The estimated yield would be most likely underestimated. To estimate the nuclear explosive yield in more accurate, it will be necessary to set aside the vertical deformation for the yield estimation.A large scale of the collapse and compaction have barred the vertical deformation from being used to estimate the nuclear explosive yield. The explosion caused uplifts of the test sites, after that a collapse and compaction were followed1. As aforementioned, the vertical deformation mapping with high spatial resolution is essential to distinguish the collapse component caused by explosion from the deformation. The OT method utilizes the intensity cross-correlation between master and slave single look complex (SLC) images. It has been widely applied to retrieval 3D surface deformation in case that the phase signal was decorrelated where a surface deformation is too steep due to large and complex deformation52. However, the traditional OT method has a trade-off between the spatial resolution and precision of measurements with respect to the offset-estimation kernel size37. To overcome the drawback, the MKOT method was applied to the 3D deformation measurement37. MKOT is the method to find representative displacements from the statistical properties of the multiple traditional OT measurements, which has a different spatial resolution and precision, by increasing kernel sizes (multi-kernel)37. MKOT can generate an optimal deformation map because the measurement noises can be successfully suppressed by averaging multiple measurements and the spatial resolution can be preserved in the procedure of determining representative value37. In addition, an error mitigation process is applied to the OT map to reduce the errors before mapping 3D surface deformation, because the OT map is affected by the topographic and ionospheric errors 41.Wang et al. (2018) reported that the horizontal and vertical deformations induced by the 2017 North Korea nuclear test were maximally about 3.5 and 0.5 m, respectively and the implosion would contribute to only local area41.Generating initial OT maps41.Conducting error mitigation42.Mapping 3D surface deformationFigure 38, (2) estimation of multiple offset measurements using multi-kernels, and 3) generation of initial azimuth and range OT maps through a 3D median filtering42.The three processing steps are applied to ascending and descending pairs. The initial OT maps are generated by the MKOT process, which consists of three main steps: (1) co-registration of master SLC image and slave azimuth common band-pass filtered SLC image 41, we need to reduce the effects in the error mitigation step. The topographic effect is caused by Earth surface’s topography. It depends on the perpendicular baseline and topographic height41. Although the perpendicular baseline of the two pairs were relatively short , the topographic effect should be corrected by using an existing DEM because the topographic variation over the study area was complex. Thus, the topographic effect was mitigated by using SRTM DEM. The ionospheric effect is commonly represented with an azimuth-directional streak pattern on OT maps as well as SAR interferograms53. Since the ionospheric effect makes the azimuth and range OT maps distorted, it should be mitigated. In this study, the ionospheric distortion was mitigated by a directional median filtering48.Since the topographic and ionospheric effects can largely degrade the OT measurement precision41.One azimuth and one range OT maps are generated from an interferometric pair via the above process. One ascending pair and one descending pair should be processed to retrieve 3D deformation, and hence at least two azimuth and two range OT maps must be generated from different geometries. In this study, we created two azimuth and two range OT maps from the ascending and descending orbits and retrieved a 3D surface deformation field from the four offset maps


In contrast, NTDs of Drosophila melanogaster and virilis CTCFs contain unstructured folded regions that form tetramers and dimers correspondingly in vitro. Unexpectedly, most NTDs are able to self-associate in the yeast two-hybrid and co-immunoprecipitation assays. These results suggest that NTDs of CTCFs might contribute to the organization of CTCF-mediated long-distance interactions and chromosomal architecture.CTCF is the main architectural protein found in most of the examined bilaterian organisms. The cluster of the C2H2 zinc-finger domains involved in recognition of long DNA-binding motif is only part of the protein that is evolutionarily conserved, while the N-terminal domain (NTD) has different sequences. Here, we performed biophysical characterization of CTCF NTDs from various species representing all major phylogenetic clades of higher metazoans. With the exception of Drosophilides, the N-terminal domains of CTCFs show an unstructured organization and absence of folded regions  TADs often also include smaller domains (sub-TADs) that are flanked by short boundary elements or longer regions (inter-TADs) that contain active chromatin and housekeeping genes. In addition, promoters, enhancers, silencers and insulators form a network of specific distance interactions that properly regulate gene transcription9. Until now, the unresolved question remains how specific distance interactions between remote regulatory elements are established and maintained through the cell cycle10.Chromosomes in the genomes of all higher eukaryotes have a highly organized architecture and consist of discrete topologically associated domains (TADs)11. It is believed that CTCF is the main architectural protein in mammals, which is responsible for the organization of TAD boundaries and distance interactions between enhancers and promoters16. CTCF was found in most of the higher eukaryotes including all studied bilateral organisms but is absent in yeast and plants18. Usually CTCFs from different organisms contain the cluster of eleven C2H2 zinc-finger domains (ZF) localized in the central part of the protein. In human CTCF, ZFs from 3 to 7 recognize specific 15 bp consensus19. The DNA-binding ZFs are the most evolutionary conserved among CTCFs that bind to similar sites in most higher eukaryotic genomes20. Moreover, it was found that even several chromatin domains controlled by CTCF are conserved in distant species21. Other ZF domains are usually less conserved and are involved in recognition of additional minor sequences22, interaction with specific RNAs23 or proteins27. The N- and C-termini of CTCF do not have structural domains and are not conserved in evolution29.Currently, the best-characterized protein involved in the organization of chromosome architecture is CTCF, which was initially found as a transcriptional repressor30. The current model suggests that the movement of cohesin complexes along chromatin31 is blocked by chromatin-bound CTCF protein, which leads to the formation of chromatin loops between CTCF sites in interphase chromosomes32. In vitro studies have shown that the cohesin complex interacts with the C-terminal domain of hCTCF31. The Drosophila CTCF homolog, dCTCF, is often associated with TAD boundaries and insulators34. dCTCF supports distance interactions between the GAL4 activator and the white gene reporter in model transgenic lines36. A novel multimerization domain was described within Drosophila CTCF (dCTCF) protein37. Deletion of this domain strongly affects the activity of dCTCF.CTCF can support distance-selective interactions between its sites, suggesting that protein-protein interactions are possibly involved in organization and maintaining long-range chromatin interactions. However, dimerization domains have not been found in hCTCF. The N-terminal domain of hCTCF was shown to be intrinsically disorderedDrosophila melanogaster raised the question about the structure of the N-terminal domain in CTCF from other bilaterian organisms. We found that CTCF in Drosophila virilis (dvCTCF) also has the dimerization domain. However, in other selected organisms from different bilaterian clades, the N-terminal CTCF domains are intrinsically disordered and unable to form dimers in vitro. Unexpectedly, the N-terminal domains from CTCF of human and several other organisms showed self-interaction in the yeast two-hybrid (Y2H) and co-immunoprecipitation assays.The existence of the N-terminal dimerization domain in Drosophila melanogaster (dmCTCF), which is essential for functional activity of the dmCTCF protein, but the first 70 residues contribute to its stability and together with 70–163 aa most likely are parts of the entire protein domain37. In Drosophila genus, alignment of the N-terminal regions of CTCFs showed a moderate level of homology with a few conserved sequence blocks in the interval of 1–163 aa according to dmCTCF sequence  of CTCF from Drosophila virilis (dvCTCF), which has the comparatively low sequence (49%) homology to dmCTCF in 1–163 interval  from dvCTCF  and dmCTCF  were expressed in bacteria and tested for dimerization using size-exclusion chromatography (SEC) Fig.  and crosers Fig. . Becauseers Fig. . This obTo further assess the oligomeric state and check the monodispersity of the purified dmCTCF NTD sample, we used Dynamic Light Scattering (DLS). The size distribution of both samples contained only one narrow peak. Estimated hydrodynamic radius (Rh) value varies in range 4.4–4.6 nm and corresponds to molecular weights of 110 kDa. However, DLS calculations of molecular weight as well as SEC are highly sensitive to the shape of the molecule, which often leads to the overestimation of protein molecular weight, so the value of 110 kDa corresponding to hexamer could result from multimerization as well as from the presence of unfolded regions.Drosophila CTCF NTDs has overall globular structure formed by unfolded regions, which explains why the SEC profile is heavier than could be expected for globular dmNTD tetramer or dvNTD dimer.To determine the correct oligomerization status of NTDs, we used a Small-Angle X-ray Scattering (SAXS) approach. Calculated molecular weights of dmCTCF NTD were in the range of 71–83) kDa corresponding to tetramer (monomer Mw is 18 kDa) in agreement with SEC data rather than with cross-linking experiments (Table –83 kDa cDrosophila species (D. virilis and D. melanogaster) using 2D NMR spectroscopy. The 15N,1H HSQC spectra for 15N-labelled dmCTCF NTD and dvCTCF NTD were found to have similar features for both proteins  and A. mellifera  are Arthropods, both belonging to Ecdysozoa phylum of Protostomia. C. gigas (cgCTCF) belongs to mollusks, which also are Protostomes, together with annelids comprising the Lophotrochozoa phylum  is a representative of Echinodermata. The role of spCTCF in the establishment of TAD borders was shown earlier21. S. kowalewski (skCTCF) is the marine invertebrate, a representative of Hemichordata, being close to basal Chordates. This organism also displays signs of reduction38. C. intestinalis is lower Chordata. C. intestinalis (ciCTCF) genome was sequenced in 2002 and despite being about 1/20 of the human genome by size, it contains an almost complete set of genes found in vertebrates, although many organs were reduced or secondary lost39. In vertebrates, CTCF proteins are described as key organisers of chromosomal architecture. Consistent with the important role in transcription regulation, vertebrata CTCFs have high homology in all characterized representatives , basal metazoans — Porifera and Placozoa. Emergence of CTCF protein is often associated with origin of Bilaterian metazoans, but CTCF homologs were not found in flatworms, presumably due to the secondary loss. Also, CTCF is absent in several clades of nematodes29.Since the cluster of zinc-finger domains of CTCF proteins is the only domain Fig.  that exhans Fig. . The funlum Fig. . A Deutelum Fig. . S. purpves Fig. . We clon41 revealed that all of them are predicted to be mostly disordered. For subsequent biochemical and biophysical analysis, CTCF NTDs were expressed in E.coli. Unfortunately, we were unable to express in bacteria a sufficient amount of drCTCF NTD. Values measured by SEC for all proteins appeared larger than could be expected for monomeric globular form and close to expected for unfolded proteins , but SAXS data (reproduced in two measurements of independent protein preparations) suggest that assemblies with a larger volume can form under several conditions. Chemical cross-linking with glutaraldehyde and EGS along with size-exclusion chromatography were used to test possible change in oligomerization status of hsCTCF NTD induced by concentration to 10 mg/ml and freeze-thaw cycles, but still, we did not observe any detectable presence of hsCTCF-NTD multimers. SAXS is extremely sensitive to the presence of high-molecular-weight particles, so most likely, these observations could be attributed to small amounts of aggregates in samples. NTDs of D. pulex and S. purpuratus also have slightly larger molecular weight than calculated for monomer, but both are unstructured . For allot Fig.  revealedila Fig.  NTDs, shDrosophila CTCF NTDs have the unique ability to form multimers in vitro among metazoans, even in contrast to related Apis mellifera. They adopt an unusual fold with the absence of secondary structure elements.Thus, in vitro does not exclude this property of NTDs in vivo. To test this possibility, we used two different approaches. The first was a yeast two-hybrid assay (Y2H). Sequences encoding the NTDs were fused in-frame to the yeast GAL4 DNA-binding domain (BD) and activation domain (AD). Because steric hindrance can interfere with transcriptional activation in the two-hybrid system, the NTD sequences were placed at both the N-terminus (NTD-AD and NTD-BD) and the C-terminus (AD-NTD and BD-NTD) of the fusion protein.Lack of the homodimerization ability of CTCF NTDs in experiments et al.37). Here we confirmed this observation for the dm-NTD and found that other NTDs also were able to interact only in one of four tested configurations , Zw5, Pita and Zipic48.The CTCF belongs to transcription factors with an arranged array of C2H2 domains. In contrast to TFs of other classes, C2H2 proteins typically bind to 12–20 bp sequences51, expansion of different domains was observed: ZAD and SCAN, respectively, which exhibit the ability to predominantly form homodimers. It was demonstrated that homodimerization ZAD from three C2H2 proteins  determines the specificity of long-range interactions52. C2H2 proteins can also have other types of multimerization domains. For example, the C2H2 protein Opbp has the N-terminal C2H2 domain that can form homodimers and is involved in distance interactions53. It was recently shown that YY1 participates in enhancer-promoter interactions by forming oligomers54. Interestingly, YY1 contains 3 C2H2 domains at the C-terminus that are involved at the same time in oligomerization and DNA binding55. Another protein, LDB1, the Lim domain binding 1 protein, contains a dimerization domain that plays an important role in enhancer-promoter interactions in various developmental pathways58.Many C2H2 proteins have N-terminal homodimerization domains. In arthropods and vertebratesin vitro, but in most cases, they show potency to self-association in vivo. Only in the case of CTCF isolated from acorn worm  did we not observe homodimerization of N-terminal domain in vivo. Thus, most N-terminal CTCF domains keep structural and functional properties despite the non-conservation of sequences during evolution. Exceptions are CTCFs from Drosophila melanogaster and virilis (Drosophilids). Those N-terminal domains are folded in vitro despite the lack of secondary structure elements. It seems likely that such domain organization was adopted in Drosophilids, as the N-terminal domain of CTCF in honey bee is intrinsically disordered. Even in Drosophilids, the structure of N-terminal domains varies between tested species: N-terminal domain of Drosophila melanogaster forms tetramer, but N-terminal domain of Drosophila virilis forms only dimer.Here, we found that non-conserved N-terminal domains of CTCFs in all tested metazoan are intrinsically unstructured 59. However, attempts to find the dimerization domain in hCTCF that can support specific distance interactions have thus far been unsuccessful. It was shown in pulldown experiments that the C-terminal part of one CTCF binds to the C2H2 zinc-finger domains of another CTCF60, but the specificity of this interaction has not been proven. It was also found that some RNAs can interact with 10 and 11 ZF and induce oligomerization of the CTCF protein61. Because many C2H2 domains can with relatively low specificity interact with RNAs64, the involvement of RNAs in protein multimerization does not explain how CTCF can support specific distance interactions.The crucial role of CTCF in supporting specific distance interaction in mammalians might suggest the ability of CTCF to homodimerize. It was shown that hCTCF can dimerize by purification of FLAG-HA-tagged CTCF complex and in the yeast two-hybrid assayUnstructured N-terminal regions of CTCFs are a good candidate for the role of a domain that supports specific distance interactions between CTCF sites. The strength of pairing between unfolded NTDs can be easily regulated by various post-translational modifications of amino acid residues, which are crucial for effective stimulation/repression of enhancer-promoter interactions. The NTDs in CTCFs lack secondary structure and sequence similarity, therefore, making it impossible to identify such domains using bioinformatics approaches. Thus, there is a probability that unstructured domains are widely distributed at the N-terminal ends of C2H2 proteins, which, however, can only be verified experimentally. Further studies are required to understand the role of the N-terminal domains in the organization and regulation of distance interactions mediated by CTCFs.Drosophila and human CTCF proteins. For protein purification purposes, protein fragments were PCR-amplified using corresponding primers (see Table Apis mellifera) were obtained from a local apiary, oysters (Crassostrea gigas) were purchased at a local food store, and Daphnia pulex culture was purchased at a pet shop. RNA was isolated using TRIzol reagent, and cDNA was obtained with reverse transcription with oligo(dT) primer following standard protocols. For other cDNAs sources, see the acknowledgements.CTCF homologues were identified using BLAST search by similarity with zinc-finger domain of 37. Stable isotope-labelled proteins were expressed according to65 and purified using the same procedure as for native proteins. Size-exclusion chromatography was performed as described37 using Superdex 200 10/300GL columns . Expected Rs values for globular and unfolded proteins were calculated as described66. Chemical cross-linking of proteins was carried out with glutaraldehyde as described previously37.Protein expression and purification were performed using standard procedures, as described previously67. Measurements were made in 0.1 cm isolated cuvette at sample concentration of 0.05 mg/ml at 20 °C. Sample concentration was calculated from peptide-bond extinction values at 205, 206, 210 и 215 nm68.Circular-dichroism measurements were performed using Chirascan instrument . The instrument was calibrated using Camphor-10-sulfonic acid, according to69. Data processing and analysis were done with the ATSAS program suite for small angle scattering from biological molecules70. The subtraction of the buffer scattering was done manually by program subtrNc. The radius of gyration Rg of protein molecule in solution was evaluated using the Guinier approximation at small angles (s < 1.3/Rg), assuming the intensity I(s) to be equal to I0exp(−(sRg)2/3). To evaluate the maximum particle dimension Dmax, the pair-distance distribution function P(r) was generated with the program GNOM so that the Rg value of protein samples had to agree with that determined from the Guinier region in PRIMUS. The molecular mass (MM) of the protein was calculated using extrapolated I0 scattering intensity and protein standards of known Mw as described71. Low-resolution ab initio structure models of CTCF(1–163aa) protein representing the protein as an ensemble of dummy atoms were constructed by program DAMMIN. The program was used to build a compact configuration of beads inside a sphere of Dmax diameter with χ = 1.05 minimal discrepancy between intensity of experimental data and that calculated from generated model.Synchrotron radiation X-ray scattering data were collected using standard procedures on the BM29 BioSAXS beamline at the ESRF  at a wavelength of 0.099 nm. The 2D detector Pilatus1M and sample to detector distance 2.87 m were used to acquire scattering data within the momentum transfer (s) covering a range of 0.033–4.9 nm-1 (s = 4πSinθ/λ where 2θ is the scattering angle). Data collection and processing were performed in an automated manner using dedicated beamline software BsxCuBE. The samples were measured at least at two concentrations. A volume of 30 μl of sample solution was placed in a 1.8-mm-diameter quartz capillary with a few tens of microns wall thickness. Thirty consecutive frames with 1 s exposure were collected from the sample at constant temperature 277 K without observing any radiation damage (characterized by systematic deviations in consecutive scattering curves). Solvent scattering was measured to allow for subtraction of the background scattering. The data from consecutive frames were inspected, normalized to the incident beam intensity and averaged in PRIMUSDynamic light scattering (DLS) measurements were performed using an instrumentation of Dyna Pro Titan (Wyatt Technology Corporation). Light scattering analysis was performed using a laser wavelength of 832 nm, quartz cuvette of 20 µl volume, temperature controlled DynaPro instrument at 4 °C and Dynamics software. The protein samples were analysed in 20 mM TrisHCl buffer (pH 7.4), 200 mM NaCl, containing 1 mM β-mercaptoethanol and 10% (w/v) glycerol. The protein was concentrated up to 1 and 7 mg/ml and filtered prior to the measurements. Sequences of 10 sample acquisitions with 1 s time duration were collected at each concentration. The value of the solution viscosity was chosen out from the corresponding table of the instrument. The hydrodynamic radius (Rh) was evaluated by Stokes-Einstein equation from the autocorrelation function of DLS measurements following standard procedures. The average MM was estimated using default Mark-Houwink parameters for a hard sphere.15N-labelled dmCTCF and dvCTCF were prepared in 95% H2O/5% D2O, 20 mM NaCl, 20 mM sodium phosphate buffer (pH 7.0 or 6.5), and 0.02% NaN3. All spectra were recorded on Bruker AVANCE 600 MHz spectrometer (Moscow State University). For 2D NMR the SOFAST HMQC pulse program was used72. The acquired data were processed using NMRPipe73, and analyzed using NMRFAM-Sparky software74.The NMR samples in concentrations of 0.2–0.5 mM for 52.Yeast two-hybrid assay was carried out using yeast strain pJ69-4A , as described previously52.Protein extracts were prepared from S2 cells cotransfected by 3 × FLAG- and 3 × HA-fused plasmids with MACSfectin (Miltenyi Biotec). Coimmunoprecipitaion assay was described previouslySupplementary data.


Prosopis species have been studied, limited research has been conducted on Prosopis pallida  Kunth mesquite flour. This study aimed to characterize the nutritional quality and bioactive properties of P. pallida pod flour and to assess its technological performance in breadmaking as a partial replacer of white wheat flour. Peruvian P. pallida mesquite flour was found to have an appealing nutritional profile, with high contents of dietary fiber (29.6% dw) and protein (9.5% dw), and low contents of fat (1.0% dw) and carbohydrates (57.6% dw). It is a source of palmitic (12.6%), oleic (35.5%), and linoleic acids (45.8%), α-, β-, and γ- tocopherols, and contains phenolic compounds such as apigenin glycoside derivatives with proven antioxidant capacities. Extracts of P. pallida flour were also found to have antimicrobial and antifungal effects and did not show hepatoxicity. When formulated as a wheat flour replacer, increasing mesquite flour levels yield composite doughs of lower stickiness and extensibility, and composite breads of lower elasticity (p < 0.01). However, up to a level of 10%, mesquite flour significantly increases loaf volume, reduces crumb hardness, and produces a more uniform crumb of small size alveoli (p < 0.01). Considering the purpose of improving the nutritional and technological quality of wheat flour bread, the addition of P. pallida pod flour can be highly recommended.Although the nutritional profile, bioactivities, and uses of mesquite pod flour from various  Prosopis comprises 44 species of nitrogen-fixing trees that are mainly distributed in the arid or semiarid regions of America. Prosopis pods are sweet fruits formed of 70–75% pericarp  and 25–30% seeds  − at m/z 563) all presented the same pseudomolecular ion, with similar fragmentation patterns, which is characteristic of di-C glycosides. The produced ions at m/z 545 [M-H-18]−, m/z 503 [M-H-60]− and a base peak at m/z 473 [M-H-120]−, suggested a hexosyl and pentosyl as the sugar moieties. The fragment ions at m/z 353 [aglycone + 83]− and m/z 383 [aglycone + 113]− suggest that the aglycone is apigenin; thus, these compounds were identified as apigenin-C-hexoside-C-pentoside isomers. This identification is also in agreement with that reported in Prosopis pods and syrups by Pérez et al. [− at m/z 593) was identified as apigenin-6,8-di-C-glucoside (vicenin), due to the observation of the ions at m/z 473 and 353 (loss of two hexosyl moieties), which points out to the presence of a di-C-hexosyl derivative. In phenolic-enriched cotyledon flour extracts from Argentinian P. alba, Cattaneo et al. [P. nigra [P. laevigata [Regarding phenolic compounds , seven cz et al.  and Quisz et al. , in whicP. nigra , and theP. pallida pod flour exhibited antioxidant capacity. The same conclusion was arrived at earlier by Silva Pinto et al. [P. pallida pod flour. The antioxidant activity of Peruvian P. pallida flour extracts could be attributed to the C-glycosyl flavonoids, which possess proved antioxidant capacity [P. alba, sugar-free polyphenolic extracts of pods also demonstrated high antioxidant activity [50 values of 7.1 μg GAE/mL [The results of bioactivities are presented in o et al.  who, by capacity . In the activity  whereas g GAE/mL .E. coli, E. cloacae, and S. typhimurium) and two Gram-positive (L. monocytogenes and S. aureus) bacteria were selected. The results obtained revealed that the P. pallida mesquite extract was effective against all the tested organisms. Furthermore, those results suggest a selective action against Gram-negative strains. The achieved MIC values for the Gram-positive bacteria range between 0.60 and 0.40 mg/mL in L. monocytogenes and S. aureus, respectively; whereas the results for the Gram-negative bacteria are considerably lower (0.20 mg/mL). The lowest MIC value  was observed in E. coli and S. Typhimurium (MIC = 0.20 mg/mL) when compared to the positive control (ampicilin).The mesquite extract was also studied regarding their antimicrobial properties. To this end, three Gram-negative , as no activity was observed against the other cell lines  studied. Nevertheless, it also did not show hepatotoxicity in the primary cell culture  applied in this study, revealing no toxicity up to the maximum concentration studied (GI50 at >400 µg/mL). The results about the cytotoxic activity of P. pallida or P. alba pod flour extracts are scarce in literature; however, some authors showed that P. juliflora leave extracts caused a significant reduction in cell viability in various human cancer cell lines [Regarding cytotoxic effects, the pod flour of ll lines ,44.P. alba pod flour in mixture with wheat flour produced softer and less consistent doughs. The weakness of the dough network can be attributed to the dilution of the gluten and/or the presence of certain mesquite flour components, such as the galactomannans, that could modify the interaction among gluten proteins.Mesquite flour affected the properties of dough stickiness  and dougp < 0.0001) and increased the dough resistance to extension (p < 0.0001), whereas composite doughs of wheat flour T65 presented lower extensibility and higher resistance to extension than those of wheat flour T55 (p < 0.0001). Nonetheless, the effect of mesquite flour on the extensibility properties of the composite dough was less marked when combined with wheat flour T55, as some stabilization in both extensibility and resistance to extension was achieved between the substitution levels of 5% and 15%  which is known to have a very distinct composition, with higher contents of protein (8.11% db), crude fibre (3.40% db), and insoluble dietary fibre (306 g/kg db), and lower contents of fat (0.77% db) and total sugars (485 g/kg db) than those of P. alba pod flour  [These findings may not be comparable with the present study since the mesquite flour used was obtained from a different species (ctively) .P. pallida seeds [p < 0.0001; Loaf volume is a parameter that reflects both gluten network quality and matrix capability to retain the gases formed during fermentation. The fact of gluten content being diluted by the addition of mesquite flour is counteracted by the action of the galactomannans of high molecular weight  present in Peruvian da seeds , which eAll crumb texture parameters were affected by the addition of mesquite flour. Regardless of the wheat flour type, replacing them with 5% or 10% mesquite flour produced softer crumbs than the control (0%), while at a substitution level of 15%, the crumb hardness increased to the same level of the control . This wap < 0.0001), which is a negative effect since less cohesive crumbs are more susceptible to disintegration. In mixture with wheat flour T65, mesquite flour added up to a proportion of 10% did not significantly change crumb springiness, although at a substitution level of 15% the loss in springiness was highly noticeable (p < 0.0001) as mesquite flour proportion increased, regardless of the wheat flour type; which suggests that the addition of mesquite flour causes loss in crumb elasticity.Crumb cohesiveness was not significantly affected  only when 5% mesquite flour replaced wheat flour T65 . All of ticeable . In mixtp < 0.0001) and the higher mean cell density (p < 0.0001)  . MesquitThe void fraction was not affected by the mesquite flour addition in the formulations with wheat flour T65. However, when replacing wheat flour T55, the addition of higher proportions of mesquite flour produced bread crumbs of progressively lower void fraction values .P. alba pod flour. They also observed that the addition of P. alba pod flour significantly affected alveoli shape, producing cells of lower circularity or compactness. However, in our study, alveoli shape was not altered by mesquite flour either in mixture with T55 or in mixture with T65, as can be appreciated from the parameters mean cell compactness (p = 0.156) measuring circularity and mean cell aspect ratio (p = 0.383) measuring elongation  affects the rheological properties of the composite dough causing lower extensibility and tenacity, and higher resistance to extension, that ultimately induce several technological properties such as low volume, less elastic/resilient, harder and shredded crumbs, increased chewiness, and compact structures of smaller size alveoli [P. pallida pod flour, except for the quality properties of loaf volume, and crumb hardness and chewiness, where the addition of this flour at substitution levels of 5%–10% exerted a beneficial effect. In this range of substitution, composite breads presented higher volume and crumbs of reduced hardness and chewiness. This may be an effect of the high molecular weight galactomannans and globular proteins present in P. pallida pod flour, which counteracted gluten dilution by forming a gel-like structure with increased gas entrapment capacity [P. pallida pod flour’s ability to reduce crumb hardness as well as the activity of its extracts against some species of fungus and Gram-positive bacteria, further studies should be conducted on the potential application of P. pallida pod flour as a natural ingredient or additive to extend shelf life of bread.Many studies have reported that replacing even a low percentage of white wheat flour with flours of legumes such as chickpea, soy and mesquite  and protein (9.5% dw), and low contents of fat (1.0% dw) and carbohydrates (57.6% dw). P. pallida flour is a source of palmitic, oleic, and linoleic acids, and tocopherols of the α, β, and γ isoforms, and contains phenolic compounds, such as vicenin II, isoschaftoside, and schaftoside, with proven antioxidant capacity. Extracts of P. pallida flour were found to have antimicrobial effect with minimum inhibitory concentrations lower than 0.30 mg/mL against E. coli, S. typhimurium, A. ochraceous, P. ochrochloron, and P. funiculosum; no toxicity up to the maximum concentration studied (GI50 at >400 µg/mL). In a future study, the endurance of bioactivities after the bread making process will be assessed.Pod flour of Peruvian P. pallida pod flour affects most of the technological quality attributes of bread, probably due to the presence of globular proteins, fibre, and high-molecular-weight galactomannans that interfere with gluten network development. When formulated as a wheat flour replacer, increasing proportions of mesquite flour produce composite doughs of lower tenacity, stickiness and extensibility, and composite bread of lower cohesiveness and elasticity. However, up to a substitution level of 10%, the addition of mesquite flour significantly increases loaf volume, reduces crumb hardness, and produces a more uniform crumb aspect consisting of more alveoli of small size. Considering the purpose of improving the nutritional and technological quality of white wheat flour bread, the use of P. pallida pod flour is highly recommended.The addition of


In addition, the blackberry seed flour extract showed capacities for anti‐inflammation and antiproliferation by suppressing LPS induced IL‐1β mRNA expressions in the cultured J774A.1 mouse macrophages and the proliferation of LNCaP prostate cancer cells. The results suggest potential health benefits and further utilization of blackberry seed flour as functional foods.The blackberry seed flour was cold‐extracted using 50% acetone and examined for its phytochemical composition and health‐beneficial properties including in vitro gut microbiota modulatory, free radical scavenging, anti‐inflammatory, and antiproliferative capacities. Among identified thirteen components of blackberry seed flour extract through UHPLC‐MS analysis, sanguiin H6 was the primary component and followed by ellagic acid and pedunculagin. For health‐beneficial properties, the blackberry seed flour extract increased the total number of gut bacteria and shifted the abundance of specific bacterial phylum, family, or genus. The extract had RDSC, ORAC, HOSC, and ABTS Blackberry seed flour is a by‐product from oil processing. Investigation of the health‐beneficial components and properties of the blackberry seed flour can lead to its potential utilization in nutraceuticals and functional foods and add value to oil manufacturers and the blackberry producers while reducing environmental contaminations. This study identified the chemical composition of blackberry seed flour using its extract and evaluated potential health‐beneficial properties including in vitro gut microbiota modulatory, free radical scavenging, anti‐inflammatory, and antiproliferative capacities. Food industry typically removes seeds when processing blackberries  and Evergreen (Rubus laciniatus) blackberry seeds had significantly greater free radical scavenging capacities compared with whole blackberry or blackberry without seeds . 2,2′‐Azinobis (2‐amidinopropane) dihydrochloride (AAPH) was purchased from Wako Chemicals . Precellys lysing kits were purchased from Bertin Technologies . 6‐Hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox), 10 ml Pyrex screw‐cap tubes, 37% Hydrochloric acid (HCl), 10 M sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), and ellagic acid were purchased from Sigma Aldrich . Lysogeny broth (LB) was purchased from Quality Biological™ . Iron (III) chloride, fluorescein (FL), 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt (ABTS), DEPC‐treated water and nuclease‐free water, and 30% ACS grade hydrogen peroxide were purchased from Thermo Fisher Scientific . cDNA Synthesis kit was purchased from Agilent Technologies . J774A.1 and LNCaP cells were purchased from American Type Culture Collection . DMEM, RPMI1640, FBS, penicillin, and streptomycin were purchased from GIBCO . QIAamp DNA Mini Kit was purchased from Qiagen . TRIzol reagent was purchased from Invitrogen Life Technologies . TaqMan Fast Universal PCR Master Mix, TATA‐binding protein (TBP) and interleukin‐1beta (IL‐1β) primers, and SYBR®Green Real‐Time PCR Master Mix were purchased from Applied Biosystems .2.2Blackberry seed flour (10 g) was cold‐extracted using 25 ml of 50% acetone at room temperature. Sonication was applied for 1 min, and extraction was performed three times . DPPH radical solution was prepared in 50% acetone. Trolox was used as the standard. The final reaction mixture contained 0.1 ml of the blackberry seed flour extract, Trolox standard or 50% acetone (the control), and 0.1 ml 0.2 mM DPPH solution. The absorbance was read at 515 nm every minute for 40 min. The relative radical scavenging capacity (RDSC) was calculated from the area under the curve and reported in micromoles of Trolox equivalents/g of dry flour (μmol TE/g).The DPPH radical scavenging capacity was evaluated according to the laboratory procedure  was used to perform the HOSC assay previously described , 30 μL of the solvent, standards, or sample, 60 μL of FeCl3, and 40 μL of 0.1990 M of H2O2 were used as an assay mixture. The concentration of the extract was 40 mg flour equivalents/mL in the reaction mixture. The fluorescence was recorded every 2 min over 4 hr. For excitation and emission, the wavelengths of 485 and 520 nm were used. The HOSC was quantified using the area under the curve and expressed relative to Trolox as μmol TE/g of the dry flour samples.A fluorescent probe equipped Victor2.7−8 M FL was combined with 30 μL of sample, standard, or solvent in a 96‐well plate. The plate was heated at 37°C for 20 min in a Victor3 multilabel plate reader . Twenty‐five microliter of 0.36 M AAPH was added to each well, and the fluorescence of the mixture was recorded every 2 min over 2 hr at 37°C. The blackberry seed flour extract's initial concentration was 43 mg flour equivalents/mL in the reaction mixture. For excitation, 485 nm wavelength was used and for emission, 535 nm was used. The ORAC value was reported as μmol TE/g of the dry flour samples.The oxygen radical absorbing capacity (ORAC) value was determined following a previously reported laboratory procedure  was delivered to the medium and incubated for 4 hr. After 4 hr, RNA was isolated from the macrophages and cDNA was synthesized using the cDNA synthesis kit. Real‐time PCR was performed using ABI Prism 7,000 Sequence Detection System using TaqMan Universal PCR Master Mix. For primers, TBP was used as a control and an IL‐1β as an inflammatory response. The PCR amplification parameters are as follow: 46 amplification cycles, 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min . In response to the blackberry seed flour extract delivery, the extract was dissolved in DMSO (1:10 v/v). In a final mixture, the vehicle, 1% DMSO, was delivered for the treatment. For cell proliferation calculation, An ATP‐Lite 1 step kit  was used. The Victor3 plate reader  was used to determine the emitted luminescence. Media were changed daily, and data were collected at five different time points including 0, 24, 48, 72, and 96 hr. All experiments were performed in triplicate.The antiproliferative capacity was evaluated using the earlier laboratory procedure . For the value comparison, multiple t tests or a one‐way analysis of variance (ANOVA) (p ≤ .05) followed by a post hoc test (Tukey's test) was used.For statistical analysis, PRISM8 software from GraphPad was used. Each data point was obtained through means ± standard deviation  hexoside, pedunculagin isomers, galloyl‐HHDP‐hexoside, procyanidin B1, sanguiin H6 isomers, kaempferol‐3‐glucoside, ellagic acid, and ellagic acid derivatives were identified with sanguiin H6 as the primary compound  spontaneously rearranges into the water insoluble ellagic acid. Therefore, to quantify the ellagitannins found in blackberry seed flour extract, ellagic acid was used as the standard. In the current study, the total ellagic acid concentration after hydrolysis was about 3 mg/g of the dry seed flour Table . Previou3.2In this study, the blackberry seed flour extract was able to enhance gut bacterial growth in a time‐dependent manner Figure . PreviouBacteroidetes phylum by two‐fold compared with the control  increased about nine‐fold in aged mice compared with the young mice  mice compared with wild‐type mice , Shigella spp., and Cronobacter spp. , induced by lipopolysaccharides (LPS) compared with the LPS stimulated control J774A.1 mouse macrophages Figure . Therefo3.5The blackberry seed flour extract at an initial treatment concentration of 0.4 mg flour equivalents/mL suppressed the growth of LNCaP prostate cancer cells after 48 hr . Previously, it has been reported that ellagic acid treatment induced a marked increase in DNA damage in LNCaP and DU145 prostate cancer cell lines in a dose‐dependent manner (Vanella, Barbagallo, et al., Ellagitannins and ellagic acid also had antiproliferative capacities in liver, skin, esophageal and oral, cervical, lung, breast, and colon cancer cell lines (Ismail et al., 4The current study identified the chemical composition of blackberry seed flour and evaluated its potential health‐beneficial properties including gut microbiota modulatory, free radical scavenging, anti‐inflammatory, and antiproliferative capacities. The presence of ellagitannins and ellagic acid in blackberry seed flour and their potential health‐beneficial properties suggest the potential value‐added utilization of blackberry seed flour in enhancing human health.None declared.This study does not involve any human or animal testing. Click here for additional data file.


Bovine respiratory disease (BRD) cause important health problems in all cattle husbandry systems. It contributes substantially to the use of antimicrobial substances and compromises animal welfare and the sustainability of the cattle industry. The existing preventive measures of BRD focus at the individual animal or herd level and include vaccination, mass treatment with antimicrobials and improvement of the animal's environment and general health status. Despite progress in our understanding of disease mechanism and technological development, the current preventive measures are not sufficiently effective. Thus, there is a need for alternative, sustainable strategies to combat the disease. Some of the primary infectious agents in the BRD complex are viruses that are easily transmitted between herds such as bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV). This conceptual analysis presents arguments for combatting BRD through improved external biosecurity in the cattle herds. As an example of a population-based approach to the control of BRD, the Norwegian BRSV/BCoV control-program is presented. The program is voluntary and launched by the national cattle industry. The core principle is classification of herds based on antibody testing and subsequent prevention of virus-introduction through improved biosecurity measures. Measures include external herd biosecurity barriers and regulations in the organization of animal trade to reduce direct and indirect transmission of virus. Improved biosecurity in a large proportion of herds will lead to a considerable effect at the population level. Positive herds are believed to gain freedom by time if new introduction is avoided. Vaccination is not used as part of the program. Dissemination of information to producers and veterinarians is essential. We believe that reducing the incidence of BRD in cattle is essential and will lead to reduced antimicrobial usage while at the same time improving animal health, welfare and production. Alternative approaches to the traditional control measures are needed. Bovine respiratory disease (BRD) is a worldwide health concern in cattle and is one of the most common diseases in calves and young stock in all production systems. The disease is multifactorial and develops in complex interaction between factors associated with the host, the pathogens and the environment. The existing preventive measures therefore include a wide range of strategies. Despite advances of newer and better therapeutic and preventive medications, as well as efforts to improve management and optimize the environment to prevent BRD, the morbidity and mortality rates have not declined. A recent review of evolving views on BRD control measures concludes that blanket vaccination and mass treatment provides inconstant control for BRD and highlights the need to reappraise the use of these measures . Our queThis conceptual analysis presents arguments for combatting BRD at the population level through improved external biosecurity in cattle herds. The rationale for such a program will be given by describing the current impact of BRD, the effect of the current preventive measures and the likely effect of additional biosecurity improvements. Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV) are two important causative agents in BRD. The Norwegian control program for BRSV and BCoV is presented as an example of a novel and alternative strategy to prevent and reduce BRD. Challenges of such a program and relevant differences between Norway and other areas are also discussed.BRD is a common disease in cattle worldwide, both in feedlots and non-feedlot husbandry systems , 3. In UBRD has negative effects on the animals' life and the producers' economy. It is a major cause of morbidity, mortality and economic loss in both the beef and dairy cattle industries , 8. FataAntimicrobial usage in animals may affect both public health and the environment . BRD andLivestock contribute to the total human-induced greenhouse gas emissions, with cattle production accounting for the majority (60%) of the livestock sector's emissions . PracticFor BRD, the severity of clinical signs, the high incidence of chronic cases, and the high mortality and morbidity estimates underscore the importance of limiting BRD to improve animal health and welfare. Freedom from disease is a fundamental aspect of animal welfare.The multifactorial nature of BRD and the global differences in production systems of beef and dairy cattle have led to a variety of prevention strategies. Common for all strategies are attempts to either improve the animal environment, strengthen the general health and host immunity and/or minimize animal exposure to the relevant pathogens. Vaccination and preventive antimicrobial medication are the most common preventive measures, with the aim to keep a low infection pressure and/or helping the host to combat infection. All preventive measures focus on the single animal or herd as the unit of interest.Mannheimia haemolytica has been found in cattle after metaphylaxis and treatment for BRD  is a co-operative owned by the producers and 96% of the dairy herds report to the Norwegian dairy herd recording system, where membership is voluntary . This prTrichophyton verrucosum has nearly been eliminated due to an eradication program that combined vaccination and zoosanitary measures  and feces (BCoV) can be used for antigen detection or for genome detection by RT-PCR . AntibodThe herd-sensitivity and herd-specificity of a diagnostic test is influenced by the basic performance of the test, the within-herd prevalence and the number of animals tested. Misclassification can arise as a result of imperfect test performance or changes in status after testing. Imperfect test performance could also be due to a suboptimal test-regime with regard to which, and how many, animals are tested.The interpretation of testing will depend on the age and number of animals sampled. Bulk tank milk serology can provide an estimate of herd-level seroprevalence of BRSV and BCoV . The metThe recently launched Norwegian BRSV and BCoV control program is presented as an example of a national level control program based on systematic improvement of external biosecurity at the herd level. The program contains no vaccination or mass-treatment. A brief description of cattle production in Norway is included for context, followed by an outline of the chosen method for herd classification and the applied biosecurity measures.Milk production in Norway is extensive and based on small, mostly family run-farms. The number of dairy herds is around 8,300, with an average herd size of 27 cows in 2018. For members of the Norwegian dairy herd recording system, production data is available to advisors and veterinarians. Many producers rear their own heifers and keep bull-calves for slaughter, which means that young stock and adult cows are often kept in the same or nearby facilities. The number of beef herds is 3,600. These are predominantly suckler-cow herds with an average number of 23 cows, which rear their calves until slaughter . There iIn a nationwide study of 134 randomly selected Norwegian dairy herds, Gulliksen et al.  found 31The control program for BRSV and BCoV was initiated by a joint cattle industry and launched in Norway in 2016. The goal of the program is to reduce the occurrence of BRSV and BCoV in the national cattle herd. A key feature is to classify all herds according to BRSV/BCoV antibody status (sero-positive or -negative) and protect animals in both positive and negative herds from infection through herd biosecurity measures. Vaccination or antimicrobial treatment is not included in the implementation plan, and vaccination against BRD is usually not practiced. Knowledge of herd status is assumed to motivate producers to implement the recommended measures for prevention of virus introduction. Furthermore, sero-negative herds who can document specific additional biosecurity measures are eligible for financial incentives.The costs of the Norwegian control program are shared between producers and the industry. The dairy industry financed the initial screening of dairy herds and the meat industry financed testing of beef herds. After that, the cost of one testing per year per herd is covered by the industry. A project leader is employed by the industry partners and responsible for information flow and education of veterinarians, producers and others within and outside the organizations.The principle of herd classification in the control program is illustrated in All samples are tested with a new multiplex immunoassay for BRSV and BCoV antibodies . The sensitivity and specificity for the bulk tank application of the test have been estimated to 94.4 and 90.6 for BRSV and 99.9 and 93.7 for BCoV, respectively  and indirect (people and fomites) routes. All producers are encouraged to avoid live animal contact between negative and positive herds by purchasing animals or sharing pastures with animals only from sero-negative herds. Live-animal trade is organized by the producer organizations, both for replacement animals and for animals shipped to slaughter. Since the launch of the control-program, separate transport vehicles have been used for animals from negative and positive herds. Farmers are encouraged to build suitable loading areas for shipment of live animals. Furthermore, improved external biosecurity is encouraged by implementing restricted human access into herds. There is a legal requirement to provide sluices where veterinarians, AI technicians, advisors, claw trimmers, service people and others can change to protective clothing and footwear provided by each herd. Advisory support from the program is provided to ensure a feasible design of these sluices. In general, the advice is for the herd to provide clothing and footwear for visitors, washing facilities with cold and hot water and suitable storage areas for equipment.To encourage compliance with the control program, herds can acquire a “Healthy herd status” by fulfilling a set of specific criteria. These criteria include having a sero-negative status for both BRSV and BCoV. In addition, the herd needs a veterinary certificate confirming high external biosecurity through the implementation of a physical barrier sluice. A loading-area for shipment of live animals to and from the herd is also required, to enable the truck driver to access the animals without entering the barn. A “Healthy herd” status is rewarded with an increase of approximately 10% in price when selling young-stock and breeding animals.A final measure to be mentioned is the establishment of a “hot-line,” where producers report episodes of diarrhea or respiratory disease by phone. This is done to enable rapid discovery of possible outbreaks, and a notification leads to warning of relevant personnel  such that necessary precautions can be taken to avoid further disease transmission and increase the vigilance in the area.We have presented arguments for biosecurity-based control of BRD and outlined the ongoing Norwegian control program for BRSV and BCoV. We argue that successful population-level disease control is possible through external herd level biosecurity measures but that several conditions must be met.Generally, the requirements for initiating a control program will differ according to biological factors , possible control measures , availability of technical tools  and socioeconomic considerations . LindberThe first element, biosecurity, is the primary focus in the control program. The aim is to reduce risk of introduction of virus both through live animals, people and fomites. A critical point is the efficacy of the recommended protective measures. This may differ according to management system and herd structure. For example, large herds have been shown to have more visitors and thereby more indirect contacts compared to smaller herds . This caM. bovis and IBR, it is probably wise to focus on these pathogens first. However, the preventive measures will generally have positive effect on the transmission of many other infections.The effect of the recommended protective measures also depends upon the compliance to these, where the motivation among stakeholders, veterinarians and producers is crucial. It is also an ongoing measure that needs to be nourished over time. Basic education, as well as a continuous flow of updated information, is necessary. Knowledge about the occurrence of the infections is useful to motivate action. The impact of BRD is well-recognized among farmers and veterinarians in Norway, and they are usually aware that BRSV and BCoV are the primary pathogens, and that BCoV also causes winter dysentery. This probably makes it easier to motivate the producers for control of BRSV and BCoV in Norway compared to countries with other Specters of BRD pathogens. For BRSV and BCoV, the documented varying prevalence, and presence of negative herds in high-prevalence areas , 59, shoThe second element, elimination of virus from infected herds, receives little attention in the program as self-clearance is regarded likely. This is probably more effective in small herds, and the small average herd size in Norway is therefore an advantage. In larger herds, naïve cattle in sufficient numbers might be available all the time, and both acute, subclinical infections and possible persistent infections are more likely. Altogether, control might be more challenging in areas where herds are larger, and more intense monitoring might be necessary. Nevertheless, biosecurity-based control might still succeed if new introduction of virus is avoided, as it will most likely be a question of time before virus cease to circulate also in larger herds.The third element, monitoring of progress, is based on the feasibility of the classification of herds, and the frequency of the testing. There is a need for herd-level diagnostic tools that accurately classify the herds in a cost-efficient manner. Serological investigations will result in an overestimation of prevalence, as earlier discussed. In the Norwegian test-regime the small average herd size might cause few first-parity cows or calves to be available, consequently reducing the herd-sensitivity. The within-herd prevalence is to some degree unknown and probably variable between herds, and within groups in the herds, which further complicates the matter. In the control program, the test-result is valid for a full year. The probability of virus introduction after classification is considerable, particularly in herds that purchase animals. An updated herd-classification based on the combination of bulk milk tank testing, herd size, information on animal movements and geographical location has been shown to provide a more accurate estimate of herd status  and coulAltogether, herd size influences all the three fundamental elements discussed here. It is also where the Norwegian situation differs considerably from most European countries. Our average dairy herd comprises 27 cows and suckler-cow herds 23, and there is an absence of feed-lots as well as few and small fattening herds. In addition, herd size might also influence the time until a new infection is detected. In Norway, the number of animals tested is the same regardless of size. In herds with many animals a control program with a more intense diagnostic test regime regarding both number of animals tested and frequency of testing, might be necessary. Herd sizes are increasing in Norway, which coincides with an increase in the recorded number of infectious diseases . InfectiStakeholders and producers are obviously concerned with the costs related to a control program; is it worth it? The financial losses due to BRSV and BCoV in Norway were analyzed by the industry prior to onset of the program. This included the available knowledge of the viruses' effect on BRD and winter dysentery, and the costs of running a control program were weighted against the impact (not published). It was concluded that controlling BRSV and BCoV would be cost-efficient and should be prioritized. There are several uncertainties in such an analysis. In a study from France, the authors assumed that a reduction of BRD incidence between 20 and 50% was a realistic outcome to expect from improvements in farm biosecurity , but furThe situation in Norway with few transmissible and notifiable diseases highlight the large impact of BRSV and BCoV. Control of these highly contagious viruses require a systematic approach, and a cooperative culture with a common goal. Previous experience from systematic eradication and control of other diseases might have contributed to a culture for disease control through prevention and joint efforts in Norway. Successful control of BRSV and BCoV here could motivate to action also in other countries. Effects on public health is a profound reason for animal disease control. The expected benefits is considerable regarding the usage of antimicrobials and antimicrobial resistance, in agreement with the present OIE strategy (90). Another expected “by-product” of the control program is the likely reduction of infections caused by other pathogens transmitted via the same routes, both endemic and emerging pathogens. The Norwegian BRSV and BCoV control program indicates a way forward in how to achieve improved animal health and welfare.Antimicrobial resistance is a major public health threat. Growing concerns regarding the environmental impacts of livestock calls for new and innovative measures to prevent endemic diseases, and thereby improve the sustainability of the cattle industry. An alternative strategy to combat BRD is urgently needed. We believe it is both desirable and possible to control BRSV and BCoV, and subsequently reduce BRD, through biosecurity measures. The Norwegian initiative represents a new way of thinking that will likely have wider implications. The ultimate goal is improved animal health, welfare and a reduction in antimicrobial usage in the cattle sector as well as a more effective production.Development of concept was done during a walk-shop where all authors contributed ideas and input. Drafting of the manuscript was led by MS, with contributions and critical review from all authors. All authors have read and approved the final product.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.