BTB domains potentially can form homo- or heterodimers. The study examines the dimerization choice of several BTB domains and finds only one heterodimer, while all tested pairs can homodimerize. The BTB domain is an oligomerization domain found in over 300 proteins encoded in the human genome. In the family of BTB domain and zinc finger–containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site, whereas the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study, we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed cell-based binding assays with fluorescent protein–BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo- and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family. BTB  domains are protein–protein interaction domains that are found in over 300 human genome–encoded proteins  includinWe recently solved the crystal structure of one ZBTB family member, PATZ1 (ZBTB19 or MAZR), from mouse and zebrafish . This woSeveral BTB domain pairs in the ZBTB family were reported to form heterodimeric structures. These studies employ techniques that range from mass spectrometry to yeast two-hybrid screening (BioGRID database ). PATZ1,In the present study, we employ a fluorescent two-hybrid assay (F2H) in mammalian tissue culture cells to assess the homodimerization versus heterodimerization of selected BTB domains, identifying only a single pair that can stably form heterodimers. Moreover, using ImmGen data , we analThe BTB domain is found in about 1% (∼300) of the proteins encoded in the human genome. The core secondary structures of the BTB domain are well conserved, and their three-dimensional fold is strikingly similar . BTB domTo study the potential dimer formation in vitro, we setup a system to screen dimer formation of the eight aforementioned BTB domains in a pairwise fashion. We repurposed the commercially available F2H assay (ChromoTek) . For thiWe note that in the F2H assay, not all pairs of BTB domains interact with the same efficiency . For exaTo address whether the varied efficiency of homodimerization  was becaTo identify possible restrictions on heterodimer formation, we investigated the expression profiles of all ZBTB proteins in various cell types of the immune system using ImmGen data . We partBcl6-Patz1 expression was negatively correlated in dendritic, mast, basophil, and eosinophil cells . We anaG of binding for five homodimers  (6GUV) but was replaced by the R39-D76 interaction upon the construction of the missing loop model. The extended MD simulation recovers the R39-D42 salt bridge. Unlike the PATZ1 homodimer, which contains dynamic salt bridges, the homodimers of BCL6, MIZ1, LRF, and PATZ2 (1R29) and continue to do so over the course of the simulation.For each dimer pair, we ran MD simulations of 500 ns. Our analysis of the interface interactions focused on the lifetime of salt bridges (plotted as barcode graphs) that have a strong contribution in the electrostatic component of the total ∆d PATZ2)  and fourd PATZ2) . Of the d PATZ2) . The salnd PATZ2  have stand PATZ2  form intAs evolutionary conservation is correlated with structural or functional roles of amino acids, we assessed the conservation score for every residue of the BTB domain. These scores are color-coded (scored from 1 to 9) in the tertiary structure of the respective BTB domains  and are G of binding by summing the free energy of ∆Eint, ∆Eele, ∆Gsol, and ∆EvdW, based on MM-GBSA calculations derived from MD trajectories of homodimers . Calculations were restricted to the equilibrated portions of the trajectory, as shown by the boxed portions of the RMSD plots in G values. We find that although the stabilization energy per residue varied in the interval  kcal/mol, the factors contributing to this energy were from different sources for each pair of homodimers. For the intramolecular interactions in the molecules making up the dimer, for all systems analyzed, the bond stretching/bending/torsions (∆Eint) which make up the local terms were all negative, indicating that local strains were relieved upon dimerization, more so in some systems  than in others . In terms of nonbonded interactions, we found that the PATZ1 homodimer is overwhelmingly stabilized by the large favorable electrostatic interactions (∆Eele), especially those established at the interface as is also corroborated by the salt bridges formed  equal to −529.1 kcal/mol for the equilibrated conformation, averaged between two duplicate MD runs (Eint) is the least favorable. Because of low variation in the RMSD  yielded information about interface interactions based on the lifetime of salt bridges .Although the PATZ1 interface has the largest number of salt bridges among the homodimers, the PATZ1-PATZ2 and BCL6-PATZ1 heterodimer interfaces established additional salt bridges . The PATBTB domain N-terminal interactions have recently been proposed to mediate dimer stability . N-termi4U2M)  . Similar4U2M) , forming2, respectively  equal to −529.5 kcal/mol. BCL6-PATZ1 heterodimer is also a favorable construct with binding free energy equal to −470.9 kcal/mol. The MIZ1-BCL6 heterodimer is the least favorable of the heterodimers considered in this study with binding free energy equal to −1.6 kcal/mol/AA. The LRF-ThPOK heterodimer is a favorable construct with binding free energy of −1.8 kcal/mol/AA on the order similar to that of the PATZ1 homodimer. At the outset, a heterodimer is expected to form if the energy gain is lower than that expected from its homodimers. For example, for the PATZ1-PATZ2 heterodimer, an expected energy is the average from their homodimers, therefore, ca. −507 kcal/mol. We find that, the ∆G for the PATZ1-PATZ2 heterodimer is −529.5 kcal/mol, therefore ∼22 kcal/mol lower than the average energy expected from the homodimers  and PATZ2 (ZBTB24), can form heterodimers in vivo. Energetic calculations confirmed that this heterodimer could form a favorable interaction interface, predominantly because of additional stable salt bridges. Despite the similar name, PATZ1 and PATZ2 only show 26.5% identity and 42.4% similarity in their BTB domain sequence. These two ZBTB family members are structurally related, being the only proteins in the ZBTB family that have an additional AT-hook motif (binding the minor groove of adenine–thymine–rich DNA) that is thought to confer an alternative DNA-binding specificity to these proteins. In our assays, we used the minimal BTB domain consisting of 157 amino acids for PATZ1 and 133 for PATZ2, lacking the AT-hook motif. This demonstrates that the AT-hook is not necessary for heterodimer formation and that BTB domains are sufficient to form heterodimeric structures. These findings reveal that the PATZ1-PATZ2 heterodimer is as stable as the PATZ1 or PATZ2 homodimers in the cellular environment, a finding that is supported by the calculated binding free energy of these complexes. Electrostatic interactions in proteins are fine-tuned by the various niches in the cellular environment, with differences of pH or ionic strength . The domThe demonstration of definitive heterodimer formation between PATZ1 and PATZ2 now will allow the questioning of the participation of each protein in the phenotypes observed in the mutation or knockout of the other factor. For example, mutations in the Zbtb24 gene result in the methylation defects observed in the immunodeficiency, centromeric instability, and facial defect syndrome type 2 (ICF2) . Does PAWe investigated the underlying structural factors behind BTB domain dimerization to understand the basis of the homodimer versus heterodimer choice. A functional consequence of homodimer formation in various ZBTB proteins is the formation of a lateral groove that is a docking site for co-repressor proteins . AlthougFormation of BTB heterodimers would dramatically increase the combinatorial target specificity of this transcription factor family. Obviously, such heterodimer formation would be restricted by the tissue- and stage-specific expression of the individual proteins. We investigated the co-expression between the protein pairs of interest, and identified a cluster of 22 co-expressed ZBTB family genes , may impThe F2H assay we introduce in this study is built on a previous iteration that tested the interaction between the minimal interaction domains of the p53 and MDM2/MDM4 proteins . This syIn this study, we determined the driving forces that contribute to dimer stability. We find by MD simulations that all heterodimers are favorable. Different mechanisms contribute to homo- and heterodimer stability. Significantly, homo- and heterodimer interfaces are typically characterized by numerous and sometimes short-lived electrostatic interactions. Thus, evolution has favored conserving the fold which serves as a template for catering to the overall functions attributed to these systems while diverse mechanisms have been used to compensate for the variations observed in family members (siblings) introduced to enable those functions. The analysis of the energy components contributing to dimerization also paves the way to design stable BTB heterodimers particularly by engineering interface residues and limiting accessibility to degron positions. Our analysis confirms that heterodimerization among ZBTB family members is infrequent and that homodimers are preferred. Nevertheless, the absence of energetic restrictions for BTB domain-mediated heterodimers suggest that more pairs of heterodimers could possibly form, increasing transcription factor combinatorial specificity.Patz1 cDNA. The murine and human proteins differ at a single position  which is Ala or Thr, respectively. Amplified fragments (Table S1) were cloned into the pcDNA TM3.1/Myc-His(−)Bexpression vector that contained either a TagGFP cDNA with a NLS or a TagRFP cDNA with no signal. BTB cDNAs were cloned into the XhoI and NotI restriction sites for TagGFP and between SmaI and NotI for TagRFP vectors, such that they encoded NLS-tagGFP-BTB or tagRFP-BTB proteins. For C-terminal FP fusion constructs, amplified fragments encoding BTB domains with primers containing NheI and HindIII were cloned into the appropriate sites of either pAC-TagRFP  or pLC-TagGFP  plasmids (ChromoTek), from which nanobody sequences were removed, such that they encoded BTB-tagGFP or BTB-tagRFP proteins. The recombinant plasmid DNA was sequenced and transfected into Baby Hamster Kidney fibroblasts (BHK-1 cells) that were modified to contain concatemeric Escherichia coli lactose operator (Lac O) sequences inserted into a single locus (ChromoTek).The coding sequence of the BTB domain of selected ZBTB family proteins was amplified from cDNA derived from the human HCT116 cell line using Q5 High-Fidelity DNA Polymerase (NEB) (Table S1). Specifically for the PATZ1 expression construct, the BTB domain was amplified from a murine List of cloning primers.Table S1 Camelus dromedarius VHH domain cAbGFP4 (PDB structure reference: 3OGO [TM3.1/myc-His(−)B expression vector using NheI- and BamHI-digested amplicons generated from the F2H platform mixture as a template with forward and reverse oligonucleotides (Table S1). This plasmid encodes a fusion protein that has a 107–amino acid N-terminal GBP fused to a 355–amino acid C-terminal Lac I domain separated by a 7–amino acid linker. The experimental approach for using the LacI-GFP nanobody (GBP) to recruit GFP-tagged proteins to the LacO locus is well characterized  fused to a nanobody specific to GFP (GFP binding protein-GBP), derived from the ce: 3OGO ).This fucterized .2, 0.1 mMEDTA, 10 mM KCl, 0.5% NP-40) of HEK293T cells transfected with plasmids encoding Myc-PATZ1  reagent at a ratio of 1:3 (DNA:PEI wt/wt). Equal mixtures of NLS-tagGFP-BTB or tagRFP-BTB and GBP-Lac I encoding plasmids were transiently co-transfected. 24 h after transfection, adherent cells were visualized using an invert fluorescent microscope (ZEISS Axio Observer Z1) with 10–20× magnification. Excitation was performed using either an HXP 120V fluorescent light source or a Colibri7 light source with LED470 or LED-Neutralwhite (540–580 nm) and Filterset 38  or Filterset 43  for tagGFP and for tagRFP visualization, respectively. Emission was detected either using a Zeiss Axiocam 503 mono or MRc5 camera.The fluorescent two-hybrid (F2H) assay , 2012 waIn the F2H assay, GFP foci were only evident when the GBP-Lac I– and tagGFP-BTB–encoding plasmids were included in the transfection mixture. GFP-RFP colocalization was only evident when GBP-Lac I–, tagGFP-BTB–, and tagRFP-BTB–encoding plasmids were included in the transfection mixture. No foci were observed if the GBP-Lac I–encoding plasmid was omitted from the transfection mixture. Because the F2H-BHK cells were not synchronized in their cell cycle, some cells were in the S-phase and contained two tagGFP foci, resulting from duplicated chromosomes. For these instances, both foci were scored as independent events. Colocalization analysis was performed manually or by using the JACoP plugin of the Fiji software version 2.1.0/1.53c (quantified in Tables S2 and S3) . ColocalTable S2. Quantification of homo- and heterodimerization in the F2H assay . Table S3. Quantification of GFP foci containing cells in the F2H assay. P-value was used for hypothesis testing.The gene expression data of 46 of the 49 ZBTB family genes were obtained from the Immunological Genome Project (ImmGen) Microarray Phase 1 and Phase 2 datasets . Probes To retrieve homologs for each of the six BTB-domain proteins , Blast  search w4U2M-chain B). The construct cloned to obtain this crystal structure expresses a forced heterodimeric fusion protein of BCL6 (WT) and MIZ1 BTB domain sequences connected by a linker peptide. The electron density from the linker peptide is not reported in the final structure, so the PDB coordinates were used in the simulation files preparated without further modifications. The BCL6-PATZ1 heterodimer structure was created using BCL6-BTB monomer (PDB entry 1R29) and PATZ1-BTB monomer (PDB entry 6GUV). The three BCL6 residues mutated to aid the crystallization process  (2NN2) was similarly modeled to fill the missing coordinates for A2/B3 residues 66–71 with ModLoop   were bacR; C84N) . MissingR; C84N) . The LRF ModLoop . The PAT ModLoop  using BA ModLoop  using LR ModLoop  by selecMD simulations were performed in NAMD using the CHARMM36 force field parameters . The simRT ln Kd, via a linear regression; see, for example, Kds. As a result, MM-GBSA values listed in this work are used to assess the relative contributions from its individual terms. Based on RMSD calculations, we determined a time interval with the most stable conformation of each structure by calculating RMSD values over 500 ns. Continuous RMSD values within 1-Å variation were considered a stable interval used for further analysis. For each stable conformation, a coordinate file (pdb) and a trajectory file (dcd) were saved separately for monomers and for the complex (dimer) without solvent. The MD log file results obtained with NAMD were used to retrieve the energy components used in the molecular mechanics/generalized born surface area (MM-GBSA) calculations . The fred script . To calcmodelarchive.org) with the accession codes ma-olypj (PATZ1 homodimer), ma-1iskk (PATZ2 homodimer), ma-zhxm1 (ThPOK homodimer), ma-hf06e (PATZ1-PATZ2 heterodimer), ma-ql2m8 (BCL6-PATZ1 heterodimer), and ma-wrsln (LRF-ThPOK heterodimer). The primary data for imaging experiments have been deposited to BioImage Archive (accession number S-BIAD418).The BTB dimer models generated in this study are available in ModelArchive (


Purpose: Report of a successful case of endovascular recanalization of an occluded superficial femoral artery (SFA) using Fiber Optic RealShape (FORS) technology. Case Report: A 79-year-old male was referred for evaluation of multiple ischemic pretibial ulcers of the right lower extremity. Computed tomography–angiography (CTA) imaging confirmed significant stenosis of the right common femoral artery (CFA) and an occlusion of the SFA from its origin to the Hunter’s canal. The patient was treated with a hybrid surgical procedure: an endarterectomy of the CFA and SFA origin was performed combined with an endovascular recanalization of the occluded SFA using FORS technology. During recanalization, the FORS guidewire slowly twisted subintimally around the occluded lumen of the SFA, maintaining the created corkscrew shape after pre-dilation with the percutaneous transluminal angioplasty (PTA) balloon and subsequent stenting. Conclusions: FORS technology can be successfully used during recanalization of an occluded SFA without the use of fluoroscopy. The corkscrew shape formed during recanalization in this case was retained during PTA balloon pre-dilation and stenting; this potentially improves hemodynamics and thereby reduces the risk of in-stent restenosis. However, expanding patient series and longer follow-up data are needed to increase the understanding of the feasibility and effectiveness of using FORS in the treatment of peripheral arterial occlusive disease. Exposure to fluoroscopy during endovascular procedures poses risks to both the patient and the treatment team ,2,3. In A 79-year-old male was referred for evaluation of multiple ischemic pretibial ulcers (W1I3fI1) of the right lower extremity (Rutherford stage 5 classification). The patient had a history of smoking, cerebrovascular disease, hypertension, renal insufficiency, and endovascular recanalization of the right common iliac (CIA) and left external iliac artery (EIA) combined with bilateral thromboendarterectomy (TEA) of the common femoral artery (CFA). The photoelectric plethysmography toe-arm index was 0.34 and duplex ultrasound showed patency of previously placed stents in the CIA and EIA, however a flow-limiting stenosis in the right CFA and occlusion of the SFA were observed. Computed tomography–angiography (CTA) imaging confirmed significant stenosis of the right CFA and occlusion of the SFA from its origin to the Hunter’s canal.2, and the fluoroscopy time (FT) was 1046 s.The treatment team decided to treat the abovementioned vascular lesions in a hybrid surgical procedure: an endarterectomy of the CFA and SFA origin combined with an endovascular recanalization of the occluded SFA using FORS technology. The patient provided informed consent for the procedure. The CFA was approached via a standard surgical approach and the atherosclerotic plaque was removed via open endarterectomy with venous patch closure. Subsequently, the venous patch was punctured and a 6F sheath was placed over a standard guidewire. Digital subtraction angiography (DSA) was performed and confirmed the SFA occlusion . FurtherAfter the procedure acetylsalicylic acid 80 mg daily was added to clopidogrel 75 mg once daily and continued for three months after which only clopidogrel was continued. After the procedure, wounds healed within 6 weeks and duplex ultrasound after 6 months confirmed patency of the treated segment without restenosis.This report presents the first ever described case of successful recanalization of an occluded SFA using FORS technology. Using 3D visualized FORS-enabled devices, it was apparent during recanalization that the guidewire slowly twisted subintimally around the occluded lumen of the SFA. In addition, during pre-dilation with the PTA balloon and placement of the SE stents, these devices maintained the corkscrew shape formed during recanalization.2, and an FT of 557 s. Predilation treatment with DCBs, stent placement, and control DSAs accounted for the remainder of the procedural radiation exposure parameters. While FORS was used in the majority of the time to create the recanalization tract (56 min), the amount of FT was relatively lower during FORS usage (103 s) than when FORS was not used (454 s). Additionally, during FORS usage, the AK and DAP were 2 mGy and 0.82 Gy/cm2 and can be attributed to the use of DSAs used to create roadmaps and ensure the accuracy of the FORS devices during recanalization. The use of FORS technology therefore enables the practitioner to perform these types of procedures while reducing radiation exposure and thus the potential health risks associated with it, such as DNA damage and long-term health effects. Moreover, FORS technology provides feedback on the force applied to the guidewire, in addition to standard tactile feedback. Excessive force on the guidewire will impair its optical signal, exceeding the boundary conditions for reconstruction of the shape of the guidewire. Therefore, the FORS technology visualizes the entire guidewire as a dashed line rather than a solid line during the period when excessive force is applied to it  for recanalization of chronic total occlusions of the femoro-popliteal tract was first described by Bolia et al.  and aimsDuring predilatation of the recanalized SFA pathway with a PTA balloon and subsequent placement of the SE stents, these devices showed a similar corkscrew shape as the FORS guidewire during recanalization. Although the fact that the corkscrew shape is not necessary for the consequence of the FORS characteristics, this corkscrew configuration may provide hemodynamic benefits as spiral laminar flow appears to be the preferential arterial flow pattern . Since rWhile the use of FORS-enabled guidewires during recanalization provides improved 3D perception of the trajectory and recanalization could be performed with laser-light navigation instead of using fluoroscopy, there are also limitations to their use. For example, the current FORS software (Release 1) is unable to visualize a narrow looped (<6 mm radius) FORS guidewire without the use of fluoroscopy. As a result, FORS visualization of the FORS guidewire is only possible if the guidewire is advanced with the tip facing forward which may increase the risk of perforation during recanalization. In addition, only the 0.035 inch angled hydrophilic FORS-enabled guidewire with a working length of 120 cm is currently available. As a result, the FORS technology is currently not applicable when performing recanalizations below the knee. Further development of the current guidewire and increasing the number of available guidewires and catheters is therefore necessary to broaden the application of FORS.This report is the first to describe a case of a successful recanalization of an occluded SFA using FORS technology. As a result, with the exception of creating a re-entry, the recanalization was performed without using fluoroscopy. The deployed SE stents adopted a corkscrew shape similar to that of the guidewire during recanalization, potentially improving hemodynamics and reducing the risk of in-stent restenosis. However, expanded patient series and longer follow-up data are needed to increase understanding of the feasibility and radiation dose reduction effectiveness of using FORS in the treatment of peripheral arterial occlusive disease.


The Mechanistic Target of Rapamycin (mTOR) pathway integrates a variety of intracellular and extracellular signaling and coordinates many essential cellular processes, including protein synthesis and autophagy. During the neurodevelopment, mTOR acts as one of the main directors controlling the differentiation and proliferation of neural progenitor cells, neuronal morphology and migration, and synaptic plasticity  model induced by homozygous Tsc2 mutant. These results demonstrate that Tsc2 has a critical role in regulating neural development and shapes inhibitory granule cells molecular and morphological characteristics. In addition, they performed a transcriptome profiling revealing a significantly different expression in the gene networks regulating neural circuitry following the loss of Tsc2 gene. Ouyang et al. assessed GWAS data of epilepsy from the International League Against Epilepsy and gut microbiota from MiBioGen, and applied Mendelian Randomization analysis to explore the potential causal relationship among the gut microbiota, metabolites, and epilepsy. The study allows to explore the etiology of epilepsy from a new dimension. In the review articles, Girodengo et al. summarized the accumulating evidence from single-cell transcriptomics and cerebral organoid-based studies to improve our understanding of the crucial role of mTOR pathway in brain development and neurological disorders. Furthermore, they highlighted new ultra-sensitive techniques for the identification of somatic mTOR pathway mutations shedding light on the neurodevelopmental origin and phenotypic heterogeneity in mTORopathy patients. Wang et al. reviewed the current advances in imaging genetics in brain microcircuits and epileptic networks during brain development. The imaging genetics analysis accurately and efficiently integrates multidimensional datasets within a unified framework, providing a unique opportunity to generate a global vision for epilepsy to promote the molecular mechanism exploration and develop targeted treatment strategies.In this Research Topic, a diverse insight into the different areas of research aimed at better understanding of neurological disorders caused by mTOR dysregulation. The original research by In summary, the present Research Topic comprises original research and comprehensive reviews highlighting the role of mTOR signaling in neurodevelopment disorders. We hope that this topic will inspire further mechanism exploration of mTOR with the aim of fully restoring function to those neurological disorders.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


TM) system, while systemic congestion is estimated by calculated plasma volume. The type of clinical patient profile as defined by the ReDS system and calculated plasma volume remains uncertain. Methods: Hospitalized patients with or without heart failure were included in this prospective study. On admission, ReDS values were measured and plasma volume status (PVS) was estimated using their body weight at the same time. Cutoffs of ReDS value and PVS were defined at 34% and −2.7%, respectively. The association between the two parameters was assessed. Results: A total of 482 patients  were included. The median ReDS value was 28%  and median PVS was −16.4% . Of the patients, 64 had high ReDS value (and low PVS) and 80 had high PVS . The high ReDS group had a higher prevalence of clinical heart failure with a more elevated echocardiographic E/e’ ratio, whereas the high PVS group had a higher prevalence of chronic kidney disease . Four out of a total of six patients with high ReDS value and high PVS had both heart failure and chronic kidney disease profiles. Conclusion: The combination of ReDS value and PVS was able to clinically stratify the types of body fluid distribution and patient profiles. Utilizing these tools may assist the clinician in constructing a therapeutic strategy for the at-risk hospitalized patient.Background: Pulmonary congestion is quantified by a remote dielectric sensing (ReDS In patients with chronic heart failure, residual pulmonary and systemic congestion following attempts at medical optimization is associated with an increased risk of morbidity and mortality . NeverthTM, Sensible Medical Innovations Ltd., Netanya, Israel) system is a non-invasive electro-magnetic technology that quantifies the degree of pulmonary congestion within one minute of application and without needing a high level of technical expertise  × [a × (b × (lean body mass))], in which a = 1530 for males and 864 for females, and b = 41.0 for males and 47.2 for females. The lean body mass was calculated as follows: 0.33 × BW (kg) + 0.34 × [body height (cm)] − 29.5 for males and 0.30 × BW (kg) + 0.42 × [body height (cm)] − 43.3 for females [PV status (PVS) was calculated as follows: PVS (%) = [((estimated PV) − )/] × 100. Ideal PV (L) = a × BW (kg), in which a = 39 for males and 40 for females. Laboratory data including plasma B-type natriuretic peptide and transthoracic echocardiography data were obtained on admission according to the standard manner. Continuous variables were calculated as a median and interquartile range. Categorical variables were calculated as numbers and percentages. The correlation between ReDS values and estimated PVS was assessed using Pearson’s correlation. A cutoff of ReDS value was defined at 34%. Patients with ReDS values above this cutoff were classified as having clinically significant pulmonary congestion. A cutoff of PVS was defined at −2.7% . AlthougAll analyses were performed in SPSS Statistics 23.0 software , and two-tailed p values less than 0.05 were assumed to be significant.A total of 500 hospitalized patients were considered for study inclusion. Of these, 18 patients with pneumonia were excluded. Eventually, 482 patients were included in the final study cohort. Median age was 76  years old and 288 (60%) were men . Of thesp < 0.001; N = 482; Median ReDS value was 28%  and median PVS was −16.4% . These values were distributed widely A,B. Therp = 0.059 and p = 0.097, respectively; p < 0.001 and p = 0.013, respectively). They also had higher prevalence of diabetes mellitus, anemia, and hypoalbuminemia than those with a low ReDS value and low PVS . Out of all the patients, 150 (31%) had a high ReDS value and/or high PVS. They tended to have heart failure (33% versus 26%) and chronic kidney disease (71% versus 64%) more frequently than those with a low ReDS value and low PVS .Out of all the patients, 64 had a high ReDS value (and low PVS) and 80 had a high PVS  . The higOnly six patients had a high ReDS and high PVS . Of these, four patients (67%) had both heart failure and chronic kidney disease and one patient had chronic kidney disease alone. Median ReDS value was 39%  and median PVS was 2.9% . Median echocardiographic E/e’ ratio was 13.2  and median serum creatinine was 3.0  mg/dL. Several key parameters were compared between those with and without heart failure . Plasma p = 0.15) and B-type natriuretic peptide levels (p = 0.11). The PVS did not have correlation with ejection fraction (p = 0.30), but had a significant but weak correlation with B-type natriuretic peptide .Among 136 heart failure patients, ReDS values did not have any correlations with ejection fraction  group had a more elevated echocardiographic E/e’ ratio, whereas the high PVS (and low ReDS) group had a higher prevalence of chronic kidney disease. Four out of six patients satisfying both a high ReDS value and high PVS had both heart failure and chronic kidney disease.Assessment of pulmonary congestion can be challenging. The ReDS system can quantify the degree of pulmonary congestion with reasonable accuracy when compared to other modalities, including high-resolution computed tomography , right hThe methodology to estimate the PVS by using the Hakim formula, as a surrogate of systemic congestion, was demonstrated and validated by previous studies ,9. Its pNo positive correlation existed between ReDS values and PVS. ReDS values overall did not have a strong correlation with PVS. Thus, ReDS cannot be an alternative to PVS, and vice versa. In other words, the combination of both modalities may be more practical to accurately understand body fluid distribution than each alone.Patients’ clinical profiles were uniquely characterized by combination of both modalities. Patients with a high ReDS value had a higher prevalence of heart failure etiology and a more elevated echocardiographic E/e’ ratio. The elevations of intra-cardiac filling pressures due to impaired cardiac function often coincide with pulmonary congestion, though may not manifest as systemic congestion . Prior aPatients with a high PVS tended to have chronic kidney disease and a lower echocardiographic E/e’ ratio. These patients may therefore have systemic volume overload due to impaired renal function , and mayA combination of both modalities may be practical to estimate patients’ profiles and determine therapeutic strategy, instead of either modality alone, particularly at the time of admission when comprehensive clinical assessments ha not yet been completed.When ReDS values are high (and PVS is low), these patients have a likelihood of clinical heart failure with increased intra-cardiac filling pressures and will benefit from vasodilators first, followed by diuretics therapy. When PVS values are high , these patients may have volume overload due to chronic kidney disease. Diuretics may be beneficial instead of heart failure-specific therapies to address systemic congestion. When patients had high ReDS values and high PVS, they would have both pulmonary and systemic congestion probably due to heart failure and renal impairment accompanying hemodynamic deterioration. Aggressive dehydration therapy using multiple therapeutic modalities would be required.On the contrary, ReDS values and PVS were not significantly stratified by the existence of heart failure. These are not diagnostic tools. Instead, we can quantify the amount and distribution of body fluid and adjust medications by referencing their combination, irrespective of any etiologies.This study is not without limitations. It consists of a moderate sample-sized cohort collected from a single center. Our cohort included relatively more patients with chronic kidney disease compared with other studies probably due to a relatively higher age and multiple comorbidities. Our findings may not simply be adopted to other cohorts given the lack of validation thus far. Further larger-scale multi-institutional studies are warranted to validate and generalize our findings.Given the lack of any cutoffs of PVS, we used a mean value of the healthy cohort. A high PVS above the cutoff in this study may not indicate clinically significant systemic congestion. Thus, PVS may not be a good diagnostic tool thus far. The cutoff of PVS with any clinical implications needs investigation in future studies. We proposed a therapeutic strategy by referencing the combination of a ReDS value and PVS, though prospective validation of these modalities in guiding therapy needs further investigation.The combination of ReDS values and PVS was able to better classify clinical profiles often associated with either pulmonary congestion, systemic congestion, or neither. These modalities may be useful adjunctive diagnostic tools to consider when developing a therapeutic strategy.


Vibrio vulnificus is a pathogenic bacterium that causes disease in marine fish, affecting fish farming and human health worldwide. In May 2021, in the Bohai Bay region, a disease broke out in commercially farmed pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatus), causing huge economic losses. The diseased fish had skin lesions, water accumulation in their abdomens, and showed tissue and organ damage. V. vulnificus biotype 2 has been reported in eels and other marine fish, but it is less reported in pearl gentian grouper. In this study, the pathogenic strain isolated from diseased fish was identified as V. vulnificus EPL 0201 biotype 2 on the basis of physiological and biochemical characteristics and the results of 16S rRNA gene and gyrB sequencing, virulence gene detection, and recursive infection experiments. To gain a comprehensive understanding of the pathogenicity and drug resistance of this strain, whole-genome sequencing was performed. Whole-genome analysis showed that the gene map of this strain was complete. The Virulence Factor Database annotation results showed that this strain had the key virulence factor genes vvhA and rtxA, which cause host disease. In addition, this strain had genes conferring resistance against cephalosporins, aminoglycosides, tetracyclines, and sulfonamides. Antimicrobial susceptibility testing confirmed the presence of these resistance genes identified in the genome. The results of this study show that V. vulnificus EPL 0201 biotype 2 is a multi-drug resistant strain with high pathogenicity.The online version contains supplementary material available at 10.1186/s12866-022-02610-1. Vibrio disease, also known as pathogenic hemorrhagic sepsis, is a bacterial disease caused by pathogenic Vibrio species, which affects a variety of aquatic animals, including fish, shellfish, shrimp, and crabs [V. vulnificus belongs to the genus Vibrio and is a gram-negative, rod-shaped, halophilic opportunistic pathogen. It was first identified as halophilic Vibrio in 1976 [V. vulnificus in 1979 [V. vulnificus is an important pathogen of human beings and aquatic animals, and can be divided into three biotypes. V. vulnificus biotype 1 represents the first isolated subtype, which mainly infects humans [V. vulnificus [V. vulnificus infection can cause sepsis and seriously endanger human health [V. vulnificus biotype 2 was first identified in European eels (Anguilla anguilla) and was isolated from infected eels [V. vulnificus biotype 2 affect eels and other mariculture fish, especially in fish farms. In recent years, there have been many reports of diseases caused by V. vulnificus in marine fish, including grouper (Epinephelus coioides), half-smooth tongue sole (Cynoglossus semilaevis), large yellow croaker (Pseudosciaena crocea), mullet goby (Mugilogobius chulae), and gilthead seabream (Sparus aurata) [V. vulnificus biotype 2 be considered pathogens with a special ability to cause Vibrio infections in fish, suggesting the name “Piscis” for this biotype [V. vulnificus biotype 3 has only been reported in Israel and causes wound infection and bacteremia in humans [nd crabs . V. vuln in 1976  and was  in 1979 . V. vulns humans . The roulnificus , 6. V. vn health . V. vuln aurata) –14. Symp biotype . V. vulnn humans .In May 2021, a disease broke out in a pearl gentian grouper breeding pond in a marine farm in the Bohai Bay area and caused serious economic losses. The laboratory obtained dominant bacteria from the diseased pearl gentian grouper and named it EPL 0201. In this study, we isolated the pathogen; performed bioinformatics analysis, virulence gene testing, API 20 Bacterial Identification System analysis, recursive infection experiments, and whole-genome sequencing; and tested antimicrobial drug susceptibility to investigate the pathogenic and resistance mechanisms of the bacterium. Our findings provide the basis for further research on the pathogenicity of this bacterium and the development of strategies to prevent the spread of infection.Diseased and moribund pearl gentian groupers  were collected from a seawater factory farm in the Binhai New district, Tianjin City, China, where the motility of the cultured pearl gentian grouper was > 75%. The diseased fish were transferred to the Tianjin Agricultural University Key Lab of Aqua-Ecology and Aquaculture, and after anesthetization using MS-222 , a camera  was used to record their symptoms.gyrB) sequencing [gyrB amplification products were sent to Sangon Biotech  for sequencing. Nucleotide sequences were submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) [gyrB sequences was performed using the EzBioCloud database (https://www.ezbiocloud.net/) [https://www.megasoftware/) was used for phylogenetic analysis based on individual and concatenated sequences [Under sterile conditions, the gills, surface mucus, and diseased tissue of the diseased fish were observed under a microscope by wet mount, and no fungi and parasites were found. Subsequently, tissues from the spleen, kidney, and intestine of the diseased fish were taken under sterile conditions, pulverized with sterile PBS buffer, and then streaked on BHI fixed medium plates , followed by culturing at 28 °C for 24 h. Dominant uniform bacterial colonies were observed on the culture plates after incubation. A single dominant bacterial colony was selected and inoculated on the same medium to obtain a pure isolate. The pure isolate thus obtained was tentatively named EPL 0201 was stored at − 80 °C in BHI broth containing sterile glycerol at a final concentration of 15% (v/v). This bacterial isolate was gram stained, and its morphology was observed using a light microscope . The isolates were fixed in 2.5% electron microscope fixative solution for 2 h in the dark, following which the morphology was observed using a transmission electron microscope . Molecular identification of the isolate was performed via 16S rRNA gene and gyrase B  . Similarequences . DistancvcgC, vvhA, rtxA, Wza, and ompU, was detected by performing PCR with their respective specific primers listed in Supplementary Table FThe presence of some virulence genes, including vcgC is a unique virulence gene of V. vulnificus biotype 1 and has weak pathogenicity but is important for distinguishing between V. vulnificus biotypes 1 and 2 [vvhA (hemolysin A) codes for a 51-kDa water-soluble protein, which is the only cytotoxin extracellularly secreted by V. vulnificus. The V. vulnificus strains carrying this virulence factor gene are highly pathogenic [vvhA is associated with tissue necrosis in the small intestine, which is often followed by the spread of infection to the bloodstream and other tissues [rtxA (cytolysin) is the most interesting virulence gene carried by V. vulnificus biotype 2 and is highly pathogenic [V. vulnificus infection mainly depends on the synergistic effect of multiple virulence factor genes. In addition to the well-defined main virulence genes vvhA and rtxA, other virulence genes, such as Wza (capsular polysaccharide outer membrane protein) and OmpU (conserved outer membrane porin), are thought to play a role in the pathogenic process of V. vulnificus [Wza plays an important role in protecting bacteria from the host’s innate immune response and is widespread among pathogenic Vibrio spp.[OmpU can mediate bacterial adhesion to host cells and enhance the pathogenicity of V. vulnificus [ 1 and 2 . vvhA  were purchased from Qianhaiyuan Farm in Tianjin, China, and kept in a 1500-L circulating water system similar to the farm environment for 2 weeks. During this period, the fish were given commercial feed  twice daily, and the water temperature was maintained at 25 °C ± 0.5 °C with 25 ppt salinity.7–1.0 × 103 CFU mL−1) was prepared with BSMFL01 . The fishes in each experimental group were intraperitoneally injected with 0.1 mL of the bacterial suspension, whereas those in the control group were injected with 0.1 mL of 0.85% NaCl. The experimental fish were continuously observed for 7 days after injection, during which normal feeding, water changes, infection incidence, and mortality were recorded, and the LD50 was calculated using the Bliss method [Before the recursive infection experiments, the fish were randomly divided into 6 groups of 12 each. Each group was placed in an 80-L water tank. Bacterial suspension  was extracted using bacterial gDNA extraction kits . The extracted DNA was detected using agarose gel electrophoresis, and the DNA quality was analyzed using IMPLEN P300 . High-quality bacterial DNA was sent to BioMarker  and sequenced using the PacBio RSII high-throughput sequencing platform .The whole-genome sequence of the EPL 0201 strain was obtained using the GenBank non-redundant protein (NR) database . EvolutiWe used the Virulence Factor Database (VFDB) and the CAntimicrobial susceptibility testing was performed using the Kirby–Bauer paper diffusion method . The bacgyrB sequences were 1434 and 1154 bp in length, respectively, and showed 100% identity with V. vulnificus KU245729.1 and 99.86% identity with V. vulnificus EU118215.1, respectively. A phylogenetic tree was constructed using MEGA 7.0  did not show any symptoms and none died  was assembled into three chromosomes and two plasmids. EPL 0201 had a larger genome than that of the reference strain for this bacterium, from the JGI IMG/MER database. It has been reported that the larger the genome, the more likely it is to contain metabolism-related and resistance genes, providing a unique mechanism for adaptation to different living environments [vvhA, rtxA, and Wza. Related studies have shown that these genes are closely related to bacterial pathogenicity [In recent years, there have been increasing numbers of reports on complete , 44. In ronments . VFDB angenicity , 59. Thegenicity . KEGG dagenicity .TEM, sul1, tet, parE, and APH, which can confer resistance to β-lactams (carbapenems), aminoglycosides, tetracyclines, fluoroquinolones, sulfonamides, and rifampicin. This result suggests that the isolate is a multi-drug resistant strain. According to the results of the drug susceptibility test, the EPL 0201 strain was resistant to antibiotics such as β-lactams , aminoglycosides, tetracyclines, quinolones, sulfonamides, glycopeptides, and lincosamides. These results showed that this strain was multi-drug resistant V. vulnificus and confirmed the resistant genes identified in its genome. However, we found that the strain was highly sensitive to carbapenem and macrolides, a finding which may be related to the type and frequency of carbapenem antibiotics used in this area or to the weakening and loss of function caused by the mutation of resistance genes [Vibrio spp. infection [V. vulnificus infection, which should be a cause for alarm.Aquatic bacteria, which are able to adapt to their environment, usually contain several antibiotic resistance factors, which enable them to resist antibiotics that may be present in aquaculture ecosystems . Analysice genes , 62. Stuce genes . These rnfection , indicatAdditional file 1: Table F1. Primers used in this study. Table F2. Raw sequencing data. Table F3. Filtered sequencing data. Table F4. Summarized data of gene prediction. Table F5. Assembly index. Fig. S1. Electrophoresis of the DNA isolated from the EPL 0201 strain. Fig. S2. Sequencing data length distribution of the EPL 0201 train. Fig. S3. Function classification statistics of eggNOG functional genes. Note: The abscissa represents the content of each eggNOG classification, and the ordinate represent the relative content of the number of corresponding functional genes. Fig. S4. GO function annotation clasification statistics chart. Note: The abscissa represents the content of each GO category, the left of the ordinate represents the percentage of genes, and the right of the ordinate represents the numbers of genes. Fig. S5. KEGG annotation classification statistics. Note: The ordinate represents the KEGG secondary classification, and the abscissa represents the percentage. Fig. S6. Original agarose gel electropherogram. Fig. S7. pictures of naturally occurring fish. Fig. S8. Electrophoresis of the DNA isolated from teh EPL 0201 strain .


The epidemiologic and medical communities have witnessed a parallel story of HIV/AIDS and Kaposi Sarcoma (KS) unfold for the better part of 4 decades. Prior to the 1980s, KS was rare in the United States  and the In the 1990s, KS incidence sharply declined, which was attributed to a decrease in HIV/AIDS-related immunosuppression due to both the introduction of antiretroviral therapy  and how JNCICS, Suk et al. .Role of the funder: The funder had no role in this editorial.Disclosures: The authors have no disclosures.Author contributions: Conceptualization, writing—original draft, writing—review and editing: ASR, KSP.


In the Nordic countries, antimicrobial use in animals and the prevalence of antimicrobial resistance are among the lowest in Europe. The network “Nordic Vets Against AMR” organized a meeting in 2021, with key actors including representatives from universities, veterinary authorities and veterinary organizations in Finland, Norway and Sweden. This paper reflects the most important discussions on education, research, policy and future perspectives, including the experiences of these countries. It concludes that Nordic veterinarians are well placed to lead the way in the fight against antimicrobial resistance and that the sharing of experiences can support colleagues in other countries. Veterinary education must go hand in hand with research activities and continuously updated guidelines and legislation. There is also a need for postgraduate training on antimicrobial resistance and prudent antimicrobial use. The veterinary profession must, by any means necessary, protect the efficiency of antimicrobials for the sake of animal health, animal welfare and productivity, as well as public health. While restrictive use of antimicrobials is crucial, the ability of veterinarians to use this medical tool is also important for the sake of animal welfare and global food security. Antimicrobial use (AMU), here referring to the use of antibacterial drugs in animals, in the Nordic countries and the prevalence of antimicrobial resistance (AMR) are among the lowest in Europe ,2. This The network “Nordic Vets Against AMR” was initiated by veterinary faculties in Finland, Norway and Sweden, based on funding by NKJ , with the aim of allowing veterinarians in the region to share knowledge and experience of how AMR challenges can be met, and providing common ground for joint initiatives. To launch the network, a digital meeting was held in late 2021, attracting around 120 participants from the Nordic–Baltic countries . The participants represented a number of key actors, including relevant veterinary authorities, national veterinary associations and veterinary faculties, and included veterinarians with different levels and fields of professional experience. Before the meeting, information about existing legislation, voluntary guidelines and policies and the veterinary curriculum in the Nordic–Baltic countries was collected. This paper reflects the most important meeting discussions related to education, research, policy and future perspectives, including some experiences in Finland, Norway and Sweden.Improving awareness and understanding of AMR through effective communication, education and training is part of the WHO Global Action Plan on Antimicrobial Resistance . In the According to a presentation by Carmen Espinosa-Gongora , veterinIn addition, the network wanted to promote lifelong learning and a changed mindset among veterinarians on the topics of AMR and AMU. This could be achieved by sharing experiences and strengths between countries in, e.g., webinars and courses, perhaps in collaboration between veterinary faculties and veterinary associations in different countries. Inter-Nordic topics could cover, for example, One Health and global AMR perspectives, success stories of prudent AMU and infection prevention. Furthermore, veterinary education in Nordic countries could involve skill building in inter-sectoral working, as tackling AMR requires close cooperation among professionals from various fields . In addiTo tackle some of the more complex issues related to AMR and AMU, a multidisciplinary, as well as a multisectorial  approach is needed. In addition, due to already existing structures and barriers between researchers and veterinary practitioners, there are challenges that need to be addressed.To address research issues related to both AMU and AMR, a wide range of expertise is often needed—for example, researchers from life sciences and social sciences, clinicians, policymakers, advisory organizations and experts in framing research messages. To make such multidisciplinary collaborations successful, all parties must recognize their respective roles and competencies and acknowledge the time it takes to make multisectoral collaboration successful. The experiences that were shared demonstrated the need to recognize the different traditions and career-building steps within different research disciplines for multidisciplinary research to work well, for example, concerning research methodology and how research findings are published and disseminated.More research is needed within several fields connected to AMR and AMU—for example, studies providing evidence for more precise and accurate individual antimicrobial treatments of different infectious diseases, infection epidemiology and studies on the contribution of different animal sources to AMR diffusion in the community.Data availability and data sharing between different sectors within One Health are needed to effectively address complex research questions, for example, between agencies, institutes and universities. However, the data must not only be available; they also need to be of good quality and harmonized to allow their full use. There might also be issues with confidentiality and data security, but these must be resolved due to the detrimental long-term consequences of AMR. There are some open data sources, such as nucleic sequence databases and overall animal population data. However, detailed data on animal production systems, health and treatment records are only available based on specific agreements, if at all.Research funding policies vary between different countries and consist of a mixture of calls where researchers can apply for any kind of research and calls addressing specific research themes. Researchers might perceive a lack of transparency when specific research themes are developed, and it might be that such themes are more politically driven than driven by research needs. This is of particular importance when it comes to research within AMR and AMU, since threats related to those areas are not so evident from a short-term perspective and might not cause alarming headlines, compared to topics such as multicontinental outbreaks of HPAI (avian influenza virus) and the COVID-19 (SARS-CoV-2) pandemic. Research on AMR and AMU needs to be addressed with a long-term perspective and a focus on proactive One Health aspects, which is not always taken into account in the current fragmented landscape of funding.Another important issue is how stakeholders at local, regional, national and international levels can access science-based knowledge to address issues related to AMR and AMU in advisory services, policies and legislation. This is a matter of funding not only research, but also providing resources for the translation of results into science-based policies.The Nordic countries are pioneers in regulation and policymaking on AMR and AMU. All three founding countries of the Nordic Vets Against AMR network  have national strategies to combat AMR ,7,8, as Key points portraying the AMU and the legislative mentality in the Nordic countries were noted in a recent paper on the Nordic approach to bovine udder health . A commoEstonia and Lithuania have also devised national strategies for combatting AMR ,17. BothAs in all EU member states, the use of antimicrobials as growth promoters has been banned in the network’s founding countries. The EU-wide ban came after an initiative from Sweden, where antimicrobials as feed additives were banned in 1986. In addition, the routine use of antimicrobials for prophylaxis was prohibited in the core countries, even before the new EU Veterinary Medicines regulation (EU 2019/6) was developed. The legislation and voluntary guidelines in the founding countries combine to make susceptibility testing before antimicrobial treatment and the use of narrow-spectrum antimicrobials, whenever possible, part of the veterinary mentality. This has been reflected in a study that was presented by Nancy De Briyne from the European Veterinary Federation (FVE) . It appeLike all microbes, resistant bacteria—commensal or pathogenic—do not respect national borders. Thus, even if the current situation in the Nordic countries is favorable, a global perspective must be applied to the AMR issue, in animals and in humans. Hence, in parallel with the continuous efforts to reduce AMU in animals, Nordic veterinarians should share the success factors that have made it possible to rear healthy and productive animals under good animal welfare conditions with low use of antimicrobials ,20,21. IIn an EU context, where legislation related to antimicrobials is already in place, the Nordic vets could promote a veterinary service more oriented towards preventive animal health, thereby reducing the need for AMU. This may require improved veterinary skills in biosecurity and vaccinology and other disease-preventive measures, in veterinary curricula or in continuing education. Additionally, EU legislation preventing veterinarians from earning revenue linked to the sale of antimicrobials should be introduced. Globally, a requirement for access to antimicrobial drugs based on veterinary prescription only should be strived for. Nordic vets participate in the COST Action CA18217 European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which focuses on guidelines for treatment and diagnostic procedures. In this context, the European Veterinary Committee on Antimicrobial Susceptibility Testing (VetCAST) will also contribute to global standards for antimicrobial susceptibility testing of bacterial pathogens of animal origin.The Nordic countries have had surveillance programs for AMR and AMU within the human, animal and food sectors for more than two decades. These data have been utilized for policy and research purposes but, as the Nordic countries are also unique in their extensive databases for monitoring animal health, there is a potential to combine and analyze these data. In addition, new data collection opportunities provided by automatic livestock management systems and new analytical tools within artificial intelligence offer new prospects to understand the dynamics of AMR transmission from farm to fork.As AMR is driven by AMU, an obvious strategy would be to explore novel ways of reducing the occurrence of bacterial infections. The development of vaccines against bacterial pathogens is advancing, but there is a gap in the functional effectivity of some of these. In addition, research on how to optimize animal husbandry and breeding for healthy animals is needed. This includes aspects of the normal microbiota and immune system of the individual animals, as well as factors such as reducing social and other stress factors, optimizing feed contents and feeding frequency and targeting the use of disinfection routines to key points in the production operation to maintain protective microbiomes.In the Nordic countries, the national veterinary and public health authorities collaborate to complement the WHO Global Action Plan on AMR and the EU One Health Action Plan against AMR. The need for future environmental surveillance is currently being discussed among these partners. Although the environment is the most dynamic and complex sector of the three One Health pillars, the environment has, so far, often been the neglected part of AMR initiatives. From the AMR perspective, the environment is an indirect transmission route for resistant bacteria and resistance genes. More emphasis is now put on understanding the relative roles of the human, animal and environmental components in the development, transmission and persistence of AMR. There is increasing activity within the Nordic countries in terms of developing and discussing methods for environmental surveillance, particularly the use of wastewater as a reflection of the local human population microbiome, AMR determinants and drivers. A deeper understanding of the environmental impact of different AMUs  is needed to manage AMR in a true One Health manner.The data presented in the meeting indicate that veterinary students in the Nordic countries have a high level of knowledge of AMR compared to other European countries. This does not mean that the Nordic veterinary faculties can afford complacency. On the contrary, they should keep improving the relevant components of their curricula and sharing their experiences. In addition, continuous professional development activities should be conducted; collaboration between Nordic veterinary faculties may be a cost-efficient way of ensuring access to such training.Competence is a concept that integrates knowledge, skills and attitudes. Overall basic veterinary competence is currently laid down in different pieces of EU legislation, of which the Directive 2005/36/EC (amended by Directive 2013/55/EU) is crucial. Based on this legislation, the EAEVE has “translated” the requirements to the more specific list of Day One Competences; the minimum standard required for veterinary teaching and the starting point for the veterinary profession and the Nordic veterinary faculties should promote more emphasis on competences within AMR and AMU for all European veterinary students.The veterinary profession must protect the efficiency of antimicrobials to ensure animal health, welfare and productivity, as well as public health. While restrictive AMU is crucial, the ability of veterinarians to use this medical tool is also important, for the sake of animal welfare and global food security.Nordic veterinarians are well placed to lead the way in the fight against AMR; sharing their knowledge and experience can support colleagues in other countries.Veterinary education must go hand in hand with continuous research activities and continuously updated guidelines and legislation. The EAEVE accreditation should require the teaching of any national guidelines in the veterinary curricula.There is also a need for postgraduate training on AMR and prudent AMU.


Paramecium mitochondria into planar lipid bilayer membranes. Whereas the literature about structure and function of eukaryotic porins was comparatively rare during the first 10years after the first study, the number of publications started to explode with the first sequencing of human Porin 31HL and the recognition of the important function of eukaryotic porins in mitochondrial metabolism. Many genomes contain more than one gene coding for homologs of eukaryotic porins. More than 100 sequences of eukaryotic porins are known to date. Although the sequence identity between them is relatively low, the polypeptide length and in particular, the electrophysiological characteristics are highly preserved. This means that all eukaryotic porins studied to date are anion selective in the open state. They are voltage-dependent and switch into cation-selective substates at voltages in the physiological relevant range. A major breakthrough was also the elucidation of the 3D structure of the eukaryotic pore, which is formed by 19 β-strands similar to those of bacterial porin channels. The function of the presumed gate an α-helical stretch of 20 amino acids allowed further studies with respect to voltage dependence and function, but its exact role in channel gating is still not fully understood.Eukaryotic porin, also known as Voltage-Dependent Anion Channel (VDAC), is the most frequent protein in the outer membrane of mitochondria that are responsible for cellular respiration. Mitochondria are most likely descendants of strictly aerobic Gram-negative bacteria from the α-proteobacterial lineage. In accordance with the presumed ancestor, mitochondria are surrounded by two membranes. The mitochondrial outer membrane contains besides the eukaryotic porins responsible for its major permeability properties a variety of other not fully identified channels. It encloses also the TOM apparatus together with the sorting mechanism SAM, responsible for the uptake and assembly of many mitochondrial proteins that are encoded in the nucleus and synthesized in the cytoplasm at free ribosomes. The recognition and the study of electrophysiological properties of eukaryotic porin or VDAC started in the late seventies of the last century by a study of Schein et al., who reconstituted the pore from crude extracts of  The age of earth is around 4.5 billion years. Stromatolites represent presumably the oldest fossils that date back to something like 3.7 billion years . How lif2-CO2 atmosphere together with dry-wet cycles and UV light  is a bacteria-like organism, which existed before the bacterial cell lineage divided into the different kingdoms . The LUCUV light . The LUCA similar concept as described for the LUCA exists also for the LECA, the Last Eukaryotic Common Ancestor . The firvia the mitochondrial outer membrane import TOM complex into the intermembrane space using a not yet fully identified import signal, which is presumably related to the β-strands of the imported proteins WW(V is given by m-V0)nF, which is a very low energy that is needed to shift the eukaryotic pores from the open into the closed configuration.Comparison of G, at voltages between ±10 and±90mV divided by the initial conductance 0G as a function of the applied voltage for experiments similar to that shown in 0G/G was found to be dependent on the type of the cation and anion present in the aqueous solution, indicating a selectivity change when the pores switched in the closed configuration at higher voltages. The exact value for the permeability ratio of potassium and chloride was difficult to obtain because the mobility of TRIS and MES inside the pore is not known. However, because of the comparably small mobility of TRIS and MES in the aqueous phase, it is possible that Pcation/Panion of the closed hVDAC1 pore is very high (around 10) in contrast to the selectivity of the open pore where Pcation/Panion is about 0.5  were−27.4mV and+35mV, respectively. This indicated a slight asymmetry in the midpoint potential, V0, where the number of open and closed channels was balanced, i.e., 0/NcN=1. It is noteworthy that the voltage dependence of eukaryotic porin (VDAC) in the first study of Paramecium mitochondria exhibited a much higher voltage dependence because n was about 4.5 and the midpoint potentials of 0/NcN were around ±20mV  can be analyzed using nd ±20mV . In a mo of 32mV .Paramecium  was in many cases either symmetrical or slightly asymmetrical with values around ±30mV to ±40mV  of C14-labeled dicyclohexylcarbodiimide (DCCD), three mitochondrial polypeptides of approximately 9, 16, and 33kDa bound DCCD . The twomembrane . The 33kmembrane . Howevermembrane , becausemembrane . Fifty pmembrane . Water-smembrane . This ammembrane . The rolmembrane . Diafentmembrane . Its actophormia . Reconstrt porin .via the enrichment of mitochondrial particles from Paramecium mitochondria with endogenous asolectin followed by the formation of solvent-depleted membranes according to the Montal-Mueller method (The first reconstitution of eukaryotic porin (VDAC) occurred r method . For thir method . The porr method . The recr method . After ir method  or to prr method . The char method . It seemThe histogram of all current fluctuations observed with Porin 31HL showed two maxima for pore distribution . One is Similar experiments were performed with many eukaryotic porins in different research groups. Typical for the pores formed by the eukaryotic porins is an open state between 4 and 4.5 nS at small voltages. However, histograms of all current fluctuation show also pores around 2 nS, which could refer to substates of the open pore. Plant porins have a somewhat smaller conductance just a little below 4 nS at low voltage. In the case of plant porins, also a second maximum was observed at 1.5 nS. The hypothesis is that this maximum also referred to the closed state or voltage-gated substates of the mitochondrial pore.anion/Pcation. For KCl that is composed of equally mobile potassium ions and chloride, the pores are slightly anion selective. This changes remarkably, when potassium ions or chloride are replaced by lithium ions and acetate, respectively. For LiCl, the potential is more negative, whereas it gets positive for K-acetate. This result is typical for general diffusion pores similar to general diffusion pores of the bacterial outer membranes  of mitochondrial porins is cation-selective, otherwise, the relatively small conductance difference for K-MES between open and closed state and the big difference for Tris-HCl cannot be understood.The conductance of the closed state of eukaryotic porins could be evaluated from single-channel recordings extended over longer times. At voltages between 20mV and 30mV, the pores close more frequently but not too often, which means that the residual conductance associated with the closing pores could be estimated from the current recordings. This procedure allows a meaningful analysis of the conductance of the closed eukaryotic pores as it is shown in via its voltage dependence (via capacitive coupling of inner and outer membranes, in which also the folding of the inner membrane may be involved (Careful analysis of the polyanion-induced closed state of rat liver porin demonstrated that it showed an interesting analogy to the voltage-mediated closed state see . This mependence . Importainvolved .in vivo and in vitro differs considerably. Nevertheless, two early studies describe the renaturation processes of eukaryotic porins in some detail. N. crassa water soluble. In this form, the protein binds to the surface of mitochondria and blocks the import of the porin precursors. The water-soluble porin may also be renatured by treatment with low doses of detergents and needs the presence of sterols in the lipid bilayer membranes (N. crassa porin were studied in lipid bilayer membranes. Following the renaturation process of different eukaryotic porins, it seems that sterols seem to be necessary, although they may modulate the properties of the pores, in particular of plant porins (N. crassa (During the first time of research into the characteristics of eukaryotic porins, these proteins were always isolated from mitochondria. However, modern research into the properties of channel-forming proteins needs very often mass production and site-directed mutagenesis of the proteins. This is possible in the case of eukaryotic porins but it is very complicated and time consuming to bring the mutated proteins back into mitochondria. Thus, it was of interest to express eukaryotic porins in bacteria and to renature eukaryotic porins for research purposes. This was possible although translation of eukaryotic porins embranes . In factembranes . Sterolst porins . Howevert porins . On the . crassa  and stig. crassa  and ster. crassa . This meNeurospora mitochondria showed crystalline arrays of putative pores in the outer membranes if the membranes were dialyzed against low salt buffers (The presence of a voltage-dependent outer membrane pore in mitochondria was an interesting feature in research into mitochondria and attempts were made to visualize the pores. X-ray diffraction of oriented outer mitochondrial membranes from plants suggested a special location of the proteins in the membrane plane . Further buffers . Single  buffers . Analysi buffers . Many at buffers .N. crassa (Starting with eukaryotic porins from yeast  and N. c. crassa , the pri. crassa . Seconda. crassa ,b. Howev. crassa . A compa. crassa . Only ne. crassa . The phyThe 3D structure of the mitochondrial pore was for a longer time a matter of debate. Based on secondary structure predictions, electrophysiology and mutagenesis several models were developed . One modE. coli (E. coli . The mitE. coli . DespiteE. coli ,b. It isE. coli . The mosThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with right hemisphere damage (RHD) occasionally complain of difficulties in conversation. A conversation is a type of communication between the speaker and listener, and several elements are required for a conversation to take place. However, it is unclear which of those elements affect communication in patients with RHD. Therefore, we prospectively enrolled 11 patients with right hemispheric damage due to acute cerebral infarction, within 1 week of onset. To evaluate patients’ conversational abilities, we used a structured conversation task, namely, the “Hallym Conversation and Pragmatics Protocol”. The topics of conversation were “family”, “leisure”, and “other/friends”. The conversation characteristics were classified according to three indices: the “conversational participation index”, “topic manipulation index”, and “conversational breakdown index”. Patients with RHD were compared with 11 age-, sex-, and years of education-matched healthy adults. The most common site of damage in the patients with RHD was the periventricular white matter. There was no significant difference in performance between the two groups according to the conversation participation index and in the discontinuance rate assessed with the conversational breakdown index. However, patients with RHD showed a lower topic maintenance rate and higher topic initiation and topic switching rates, according to the topic manipulation index. Therefore, we explored the characteristics of impaired conversation abilities in patients with RHD by assessing their ability to converse and manage topics during structured conversations, and found difficulties with pragmatics and communication discourse in these patients. The human brain consists of the left and right hemisphere, and each is involved in the functioning of the brain with different dominance. Generally, in right-handed individuals, the left hemisphere is known as the dominant hemisphere; herein, the language function is predominant. However, patients with right hemisphere damage (RHD) occasionally complain of communication difficulties, despite preservation of normal basic language abilities . The chaA conversation is one of the ways people naturally communicate, and it can occur between two or more speakers and listeners , 6. DuriPrevious studies have examined the effects of RHD due to stroke or trauma on normal discourse, including conversations , 4, 15; However, these previous studies rarely involved structured conversations. Their results were obtained by conducting relatively free conversations with participants. Conversation tasks can be useful indicators of communication because they allow us to look at the use of language in context, better than that in procedural or narrative discourses, thereby enabling us to understand the characteristics of speech produced by the individual . HoweverAssessing brain function through stroke lesions is one of the traditionally used methods in the investigation of brain function. When a stroke occurs, the part of the brain affected by the stroke loses its function, which allows us to assume the function of the part where the stroke occurred, based on the patient’s symptoms . HoweverWe conducted a structured conversation task-based study on patients with acute cerebral infarction in the right hemisphere within 1 week of onset, in order to provide a basis for evaluating patients with right hemisphere lesions, whose language function is not impaired according to a general language assessment. The aim of this study was to compare the characteristics of conversation in patients with acute cerebral infarction with lesions in the right hemisphere with those of a healthy control group. In addition, we also describe the lesions that our patients presented with, in order to examine the relevance of these lesions in the impairment of conversational characteristics.This study included 11 patients (4 men and 7 women) whose right hemisphere was damaged due to cerebral infarction (RHD), who visited the Department of Neurology at a hospital located in Chuncheon, Republic of Korea. In addition, we analyzed patient subgroups by dividing the RHD group into those with damage only to the subcortical area  and those with damage to both the cortical and subcortical areas . Eleven additional age-, sex-, and years of education-matched healthy adults (HA) were enrolled in the study as controls. The inclusion criteria for all participants were: 1) Korean language was their mother tongue; 2) no motor speech disorders and normal hearing and vision; 3) normal Korean-Mini Mental State Examination (K-MMSE)  results In order to assess the basic cognitive abilities of the participants, we conducted the K-MMSE, which is a screening test consisting of 30 points and takes about 10 minutes to complete. The K-MMSE consists of items that determine an individual’s orientation to time and place, attention, memory, language ability, calculation ability, and visuospatial ability, with a higher score indicating a higher cognitive ability. Since depression may affect an individual’s performance on cognitive and language tests and may also be closely related to the person’s degree of participation in conversational situations, we confirmed the degree of depression of our participants with the SGDS, which consists of 15 points, with higher scores indicating greater severity of depression. In order to assess the basic language abilities of our participants, the STAND, which is made up of 20 points and takes about 10 minutes to complete, was implemented. The STAND includes subdomains of spontaneous speech, auditory comprehension, repetition, and naming. The higher the score, the higher the language ability. The EHI was conducted to identify the dominant hemisphere of the patients with RHD, and the HSQ was conducted to confirm that there were no other mental or physical problems affecting the cognitive and language abilities of our participants.According to research on elicitation methods for conversational data from clinical groups , it seemBased on such previous studies, we used the “Hallym Conversation and Pragmatics Protocol” , 36. ThiThe topics of conversation were, in sequence, “family”, “leisure”, and “other/friends”, which are all relevant to daily life. In order to facilitate the initiation of a conversation, four related photos were prepared for the “family” and “leisure” topics, and the photos were placed in separate envelopes according to the topic. The photos used in this task were not provided with the intent to force participants to look and describe exactly what they saw. Rather, based on the procedure of a previous study for conversation sampling , we usedAt the start of the session, the examiner took 10 minutes to build rapport with the participant by asking them simple questions . Thereafter, the examiner as a conversational partner explained the task instructions, and, if the participant was confirmed to have understood the task, the examiner provided two envelopes containing the pictures for the topics related to family and leisure, and the participant randomly selected one envelope (topic) first. The examiner then confirmed that the participant had seen all four photographs for each topic, set aside the photographs, and provided an opportunity for the participant to initiate a conversation. If the participant did not initiate the conversation within 3 seconds, the examiner made a comment regarding the topic, saying “So we will talk about family life/leisure activities”. If there was no response despite mentioning the topic, a related question was asked . If the participant continued the conversation, the examiner responded with neutral responses  , 42. If All the collected conversation data were transcribed within 1 week. Based on the criteria used in a previous study , unintelThe conversational participation index reflects the extent of engagement in the conversation and is based on the assumption that conversational turns include more than one utterance. Two sub-indices are used: the number of turns, and number of utterances per turn. The number of utterances per turn describes the amount of speech the participant generates in one conversation turn, and reflects how much he or she talks in general. For the latter, the total number of utterances is divided by the total number of turns.The topic manipulation index measures how often a new topic is initiated or changed during a conversation when the concept, vocabulary, and components are equally connected to the previous topic. This index includes all topics covered by researchers and participants during the completion of the dialog protocol. Five sub-indices are used: number of topics, number of turns per topic, percentage of topic initiation, percentage of topic maintenance, and percentage of topic switching. The number of turns per topic refers to the number of utterances produced per topic, and is calculated by dividing this number by the total number of topics that are discussed until the dialog protocol is completed. Topic initiation occurs when the participant speaks first when inducing a dialogue. The topic initiation rate is measured by dividing the total number of topics initiated by the total number of turns. Topic maintenance means that the participant continues the conversation by adding related content or information in response to the questions or contents of the other party. The overall topic maintenance rate is measured by dividing the total number of topics maintained by the participant by the total number of turns. Topic switching refers to the topic of conversation changing to a new subtopic that has not appeared in previous utterances while the previous turn is still ongoing. The topic switching rate is measured by dividing the total number of topic changes by the total number of turns.The conversational breakdown index assesses conversation interference. Two sub-indices are used: percentage of overlap and percentage of discontinuance. Conversation overlap occurs when one partner interferes with the other person’s words during the conversation (the participants intervene when the examiner speaks). The conversation overlap rate is measured by dividing the total number of times the topic overlaps during the conversation by the total number of turns. Conversation discontinuance is a case of failure to respond immediately to the other person’s words, delay of more than 3 seconds, abnormal long pause, or no response at all. The rate of discontinuance of a conversation is measured by dividing the total number of discontinuances that occurred during the conversation by the total number of turns. Each measurement of conversation performance is presented in To confirm the inter-rater reliability of measurements, we calculated the agreement between the data analysis of two analysts. The first analyst was the examiner (H.Y.J) with a major in speech-language pathology and more than 2 years of clinical experience at the time of the study. The second analyst was a Master’s student majoring in speech-language pathology, who was also a certified speech-language pathologist. The first and second analysts received training in conversational procedures and analytical methods. A random sample comprising 20% of the data was selected and analyzed, and the analytical reliability was found to exceed 90%. The agreement rate was assessed for the three indices, namely the conversational participation index, the topic manipulation index, and the conversational breakdown index. The rates of agreement were as follows: 1) conversational participation index, 97.17% (number of turns: 97.21% and number of utterances per turn: 97.14%); 2) topic manipulation index, 96.83% ; and 3) conversational breakdown index, 98.08% (overlap: 98.26% and discontinuance: 97.91%).Standardized T1, T2, fluid attenuated inversion recovery, and diffusion-weighted images (DWIs) were acquired for all participants at the Chuncheon Sacred Heart hospital using the same 3.0 T magnetic resonance imaging (MRI) scanner . Images were obtained in one session for all participants, and all MR images were obtained in the same orientation and slice positions. DWIs were obtained using the following parameters: axial slice thickness of 3.0 mm, inter-slice thickness of 4.5 mm, repetition time of 3980 ms, echo time of 84 ms, flip angle of 90°, and matrix size of 256 × 256 pixels.http://www.mccauslandcenter.sc.edu/mricro/; Chris Rorden, Columbia, SC, USA) Axial slices were matched to the Montreal Neurological Institute T1 template within the MRIcro software program. The lesions, drawn as regions of interest for each patient, were displayed on a common template in order to determine areas of lesion overlap.To illustrate the distribution of infarcted lesions, axial slices from DWIs of each patient were manually marked on the high-definition structural brain template using MRIcro software , topic manipulation index , and conversational breakdown index (rate of overlap and rate of turn discontinuance) between groups, the Kolmogorov-Smirnov and Shapiro-Wilk tests were used first to test for normality. The results of this analysis showed that a few measurements (rate of overlap and rate of turn discontinuance in the HA group) did not satisfy the normal distribution assumption. In addition, because the number of samples included in each group was small, the Mann-Whitney U test, a nonparametric test, was performed for all measurements. Statistical Package for the Social Sciences version 22  was used for all statistical analyses.There was no significant difference in age, sex, or years of education between the HA and RHD groups. The SGDS, K-MMSE, and STAND scores were also not significantly different between the HA and RHD groups. In addition, in terms of the impaction of the visuospatial neglect, we observed that no RHD patients had neglect symptoms through bedside observation. We also confirmed that 10 out of 11 RHD patients had no problems with the interlocking pentagon drawing item included within the MMSE. Participant information is provided in The lesion overlap maps for the RHD group generated with the MRIcro software are shown in There was no significant difference in the conversational participation index between the two groups . The datAccording to the topic manipulation index, the RHD group had a lower topic manipulation ability, a higher total number of topics, and a lower number of turns per topic than the HA group. The RHD group also showed higher rates of topic initiation and switching compared to the HA group. The topic maintenance rate was lower in the RHD group than in the HA group . The datThe conversational breakdown index showed that the RHD group had a higher rate of overlap than the HA group. There was no significant difference in the rate of discontinuances between the two groups , and no In this study, we compared the conversation characteristics in a group of patients with RHD with those in a healthy control group based on three indices—the conversational participation index, the topic manipulation index, and the conversational breakdown index. The comparison of topic manipulation index revealed that the RHD group had a lower topic maintenance rate than the HA group, but that topic initiation and topic switching rates were higher in patients with RHD. The conversational breakdown index also revealed that the RHD group showed higher rates of overlap than the HA group.We found no significant difference between the RHD and HA groups with respect to the conversation participation index, which assessed the number of turns and the number of utterances per turn. Previous studies have shown inconsistent results. Patients with RHD showed a greater number of utterances than healthy participants in one study , while iOn assessment of the topic manipulation index, we found that RHD group participants frequently changed the topic, even within their turn, when they were supposed to talk about one specific topic. Normally, in order for the speaker to maintain a topic within the conversational turn, the context of the previous as well as the next utterance must be maintained. Frequent initiation of new topics or switching of topics could be perceived as being out of context from the listener’s point of view, making it difficult to understand the context of the conversation. Additionally, since patients with RHD often provide unnecessary details, insufficient content, or broadly related but not specifically appropriate information, their speech lacks relevance . PatientWith respect to the conversational breakdown index, the RHD group had higher overlap rates than the HA group. In order to avoid overlap in a conversation, one needs to wait until the other’s turn has ended. In general, speakers may use specific utterances (syntactic or semantic) for turn endings while simultaneously using prosodic factors such as a decrease in speed or pitch , 53. TheIn a previous study using fMRI, the right hemisphere was found to be more involved in conversational topic manipulation than the left hemisphere. During a topic maintenance task, the right hemisphere homologues of Broca’s and Wernicke’s area, dorsolateral prefrontal cortex, and cerebellum were activated . AnotherWe found no significant difference between patients who had only a subcortical lesion and those who had both cortical and subcortical lesions. Although most cortical and subcortical lesions did not overlap, the periventricular white matter area was found to overlap across patients. Periventricular white matter is white matter within 10 mm of the ventricular surface , includiPatients with RHD show not only communication disorders, but also cognitive disorders , and theOur patients with RHD complained of difficulties in pragmatics and conversational discourse, but they had not previously been screened for language function, which is usually done by assessing left hemisphere-oriented language function. There exists a battery of tests to evaluate the cognitive-communication abilities of patients with RHD, such as the Mini-Inventory of Right Brain Injury-2 , the RigThis study has several limitations. First, the numerical values of the study results showed a greater standard deviation for the RHD group than for the HA group. While the type of cerebrovascular disorder and number of days after the onset of the disease were controlled for, the lesion size and site of lesion were not. As such, any such discrepancies may have affected the standard deviation of the patient group . Second,This study investigated conversational disorder in patients with RHD in the acute stage of stroke. In these patients, topic manipulation impairments and conversation breakdowns occurred. These deficiencies may be one reason why communication is difficult between them and their interlocutors in a variety of communication situations. These impairments also occurred with or without cortical involvement. Therefore, our study suggests that we should reconsider the common misconception that the language function of patients with RHD is unaffected, even if their basic language assessment is in the normal range.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.