The present study opens the possibility of applying nanostructured materials to innovative food packaging which, through nanosensory zones, change color as a function of the food pH.Colorimetric sensors developed by the solution blow spinning (SBS) technique have a rapid response to a variation in different physicochemical properties. In this study, polystyrene nanofibrous (PSNF) mats containing the bromothymol blue (BTB) indicator were obtained by SBS for the pH sensing of wine sample. The incorporation of the indicator did not promote changes in fiber diameter but led to the appearance of beads, allowing for the encapsulation of BTB. The halochromic property of BTB was retained in the PSNF material, and the migration tests showed that the indicator mats presented values below the maximum acceptable limit (10 mg dm The engineering of nanostructured materials has played an important role in the development of techniques designed to obtain nanomaterials with great potential for application in biomedicine , civil c2 production [Intelligent packaging incorporates devices that are able to monitor, in real time, the quality of the food and/or the packaging itself through responses to chemical changes, such as variations in pH ,13,14, Coduction , food froduction , or a tioduction , among oThe use of colorimetric sensors is one of the most basic and convenient analytical techniques, being easy and low-cost . The tecAmong several advantages, colorimetric sensors are attractive and are used for identification of analytes in different areas , such asPolymer filaments can be produced in two ways by electrospinning (ES) and solution blow spinning (SBS). From these methods, it is possible to obtain fibers of the sub-, micro-, and nanometric scale . However2 or N2) move in a concentric and parallel manner [In the context of polymer nanomaterials, the SBS technique is considered an innovation in obtaining versatile one-dimensional nanomaterials, especially nanofibers. In this technique, two parallel concentric fluid streams: (i) a polymer solution formed by a volatile solvent; and (ii) a pressurized gas  halochromic nanofibers integrated with the BTB indicator. The PS/BTB nanofibrous mats were investigated with respect to fiber morphology, indicator migration (dye leaching process) in aqueous media and halochromic behavior in aqueous media at different pH and exposed to acid vapors in red wine samples.5 g mol−1 was purchased from Sigma-Aldrich  and used to obtain the nanofibers. Toluene  and acetone p.a.  were used as spinning and solubilization indicators, respectively. Bromothymol blue, BTB,  was incorporated into the nanofibers to obtain the indicator mats. Phosphoric acid , monobasic sodium phosphate , dibasic sodium phosphate , anhydrous sodium carbonate , sodium bicarbonate  and sodium hydroxide  were used in the preparation of buffer solutions and for pH adjustments. The halochromic profile of the mat was studied in the presence of the following acids: glacial acetic acid P.A.  (AA), hydrochloric acid P.A.  (HCA) and sulfuric acid P.A.  (SA). For the beverage test, a Merlot dry red wine  obtained from a local supermarket was used.Polystyrene (PS) with a molar mass of 1.9 × 10−1 NaOH solution. All solutions were prepared at room temperature (25 °C) while stirring at 500 rpm. Next, the PS and BTB solutions were blended at a ratio of 3:1 for subsequent spinning at room temperature (25 °C). PS nanofibers with BTB contents of 0.05, 0.1 and 0.2%, dry weight was prepared and called PSNF/BTB-0.05, PSNF/BTB-0.1, and PSNF/BTB-0.2, respectively. Control nanofibers (PSNF control) were obtained using the same procedure in the absence of BTB.The polystyrene nanofibrous (PSNF) mats and PSNF mats with the pH indicator (PSNF/BTB) were obtained by the SBS technique. A 30 wt.% PS solution was prepared in toluene, while solutions with different concentrations of BTB  were prepared in acetone. The BTB solution pH was adjusted to 13 with a 1 mol L−1 through a 0.5 mm diameter stainless-steel needle. To promote fiber formation during the ejection process, a needle protrusion distance of 0.2 mm was used and a working distance of 15 cm (distance between the needle and the rotating collector). The fibers were gathered on the surface of a collector rotating at 420 rpm for 30 min. Room temperature and relative humidity (RH) were 25 °C and RH ≤ 55%, respectively. The samples were placed in a desiccator under vacuum for 3 days for prior subsequent analysis.The blow spinning process apparatus is represented in −1 at a resolution of 4 cm−1. The fiber characterization was performed before and after 24 h of material immersion in solutions at pH = 2.0, 6.8 and 10.7. In addition to the visual color change of the nanofibrous mat, these pH values were determined through preliminary analyses to serve as reference values for evaluating the degradation of various foods.The nanofibrous mats were characterized by Fourier transform infrared (FTIR) spectrophotometry using a spectrophotometer model Vertex 70 Bruker . The spectra were recorded in attenuated total reflectance (ATR) mode, accumulating 32 scans over a spectral range of 4000 to 400 cmScanning electron microscopy (SEM) analyses were performed using a Zeiss DSM 960  microscope. The samples were previously coated with gold using an SCD 050 Bal-Tec sputter coater . The mean fiber diameters were calculated using ImageJ software , analyzing the morphology of 100 randomly chosen fibers.w/v) AA and 20% (v/v) ethanol, respectively) according to EU Commission Regulation n. No. 10/2011 [The migration assays followed the methodology proposed by Agarwal et al.  with mod 10/2011 . The absE) between two PSNF/BTB samples subjected to the different conditions was calculated by the equation:L* is the difference in lightness between the samples, while Δa* and Δb* are the differences in the red and yellow colors, respectively, between the samples.The colorimetric analysis of the PSNF/BTB mats was performed by determining the color space coordinates of the system defined by the International Commission on Illumination (CIE), as obtained using a Minolta Chromameter CR-400 . In the CIE color space: L*a*b*, L* stands for lightness, a* represents the position between green  and red  and b* the position between yellow  and blue . In addition, the hue angle (0–360°) was evaluated to identify and quantify the changes in the color of the sensor mat  and the location of the color on the colorimetric circle. Mat color measurements were performed after 24 h of nanomaterial immersion in buffer solutions, and an average of ten readings was made for each analysis. The total color difference , hydrochloric acid P.A. (HCA) and sulfuric acid P.A. (SA) at the following concentrations: 0.0, 1.2, 2.4, 4.8, 7.6 and 10.0 gLo et al.  and Hopfo et al. . Then, abS corresponds to the relative standard deviation of the blank.The sensitivity of the nanofiber sensor to determine the presence of acid vapors was determined by the slope of the analytical curve (α) in the graph of equation ,42:(2)LOThe PSNF mats containing different BTB concentrations, obtained by the SBS technique, are shown in The increase in the BTB concentration in the spinning solution resulted in a visual change in the color of the nanofibrous mats from white to blue, and for the highest BTB concentration (PSNF/BTB-0.2), a higher intensity of the blue color was observed. To evaluate the effect of adding the indicator on the morphological profile of PSNF mats, SEM micrographs in the absence and presence of BTB were obtained, and the respective mean diameters were calculated, as shown in The morphological profiles of nanofibrous mats in the absence and presence of BTB were similar. The micrographs showed, however, that the presence of BTB at a concentration of 0.2% m/m B led to According to the literature, the use of pH indicators to obtain polymeric nanofibers usually did not result in changes in the mean diameter of the fibers or the formation of beads during spinning ,28. HoweThe addition of components to the polymer matrix during the spinning process for obtaining the nanofibers can promote changes to or the emergence of new bands in the FTIR spectra of the samples, indicating possible intermolecular interactions or chemical bonds between the components forming the nanofibrous mats . Thus, t−1 referring to the bending vibration and C−H out-of-plane bending vibration of aromatic rings, respectively [−1 was associated with the symmetric C=C in-plane vibration of the monosubstituted aromatic ring, and the one at 1583 cm−1 with the C=C in-plane stretching of the benzene ring [The pure PS polymer spectrum (not shown) did not differ from the spectrum of the PS nanofiber in the absence of BTB, indicating that the blow spinning process for fiber formation did not affect the polymer structure. In the spectrum of the control PS nanofibers, the polymer was atactic. This characteristic was indicated by the bands at 754 and 540 cmectively ,45,46,47ene ring ,49. The −1 and 1026 cm−1, corresponding to vibrations of the −SO3 group [−1 were associated with symmetric and asymmetric S−O−C stretching, respectively [−1 [−1, a narrow band appeared due to the vibrational stretching of the aliphatic C−Br bond [In the pure BTB spectrum, the characteristic bands were identified in regions near 1190 cmO3 group . The banectively . A low-ively [−1 . At 652 −Br bond .3 groups) and the hydrophobic surface of the polymer [−1  between the samples analyzed is shown in Samples of PSNF/BTB-0.2 were submerged in solutions with pH of 2.0, 6.8 and 10.7. E values obtained were higher than 5 for all sample combinations, indicating that the difference between the colors of the mats subjected to different pH values can be easily identified with the naked eye [The PSNF/BTB-0.2 (reference) mat, in contact with the acidic medium, showed a reversible change in color from blue to yellow, and the hue angle ranged from 297° to 45°. Furthermore, the Δaked eye . Furtheraked eye , that isE values were lower for the films than those for the nanofibers. The PS/BTB-0.2 films presented acid-basic halochromic capacity smaller than 5, showing that the colors of both materials belong to the same quadrant [In addition, the ability of the PSNF/BTB-0.2 sensor to respond to pH change was compared with that of a sensor formed by a PS film incorporated with the same BTB concentration . The ΔE quadrant . These ra value of 7.1, and in acid media (pH < 6) is completely protonated, exhibiting a yellow color. In basic media (pH > 8), the dye is almost completely in its deprotonated form and has a blue color [BTB is a dye with a pKue color . The BTB−1, related to C=O stretching [−1, associated with the symmetric S−O−C (thioester) vibrational stretching of BTB [−1. This result may have been caused by changes in the structure of the indicator due to protonation/deprotonation of the sulfonate and phenyl groups [The immersion of the PSNF/BTB-0.2 mat in the different aqueous media (regardless of the pH) led to the disappearance of some bands in the FTIR spectra including the band at 1703 cmretching , and theg of BTB ,58,59. Ml groups .Despite the important changes observed in the FTIR spectra of the PSNF/BTB-0.2 mat after immersion in aqueous media with different pH values, the disappearance of some bands could be associated with the migration of the indicator from the fibers to the solution, reducing the concentration of the BTB in the indicator mat and making it undetectable in the FTIR spectrum. At the same time, this phenomenon of migration has great importance in determining the application of indicator mats with foods. Thus, the migration of BTB from the PSNF/BTB-0.2 mats was evaluated in food simulation media as a function of time, and the results are shown in w/v) AA) was approximately twice that to simulator C (20% (v/v) ethanol), exceeding the maximum limit defined by the EU. Commission Regulation No 10/2011 (10 mg dm−2) [−2, the proposed system can be applied with hydrophilic foods containing up to 20% alcohol content and possessing a significant amount of organic ingredients that make the food more lipophilic [BTB migration from the mats occurred for the two simulators evaluated, and after 1440 min (24 h) of migration, the BTB phase equilibrium was reached for both simulators. At equilibrium, the migration to simulator B (3% (mg dm−2) . Therefopophilic .The high migration of the dye to simulator B can be explained by the lower stability of the PS-BTB interaction and/or the greater solubility of the compound when exposed to acidic media, thus, inducing the release of the BTB incorporated in the nanofibers.The wine identity and quality standard (IQS) depends on the sugar content , the type of grape  and the color , promoting a multifaceted definition of wine quality ,40. The Other acids are found in the food industry. HCA is widely used in the hydrolysis of starch and proteins . SA, wheThe results show a high sensitivity of the nanofiber mat to the volatile acid compounds of the sample, indicating a color change from blue to yellow. This color change can be explained by the acid-base equilibrium discussed in −1) was greater than that observed for CA, and SA, with 4.79 and 3.79 L g−1, respectively. The LODs of the components were calculated, obtaining values of 0.53, 1.24 and 1.60 g L−1. Most likely, the method of detection used in our methodology, i.e., through contact between the mat and the vapor generated by the sample, favored the sensing of AA. This conclusion is due to AA being a volatile acid, unlike the others that have low vapor pressures when in aqueous solution. Most likely, in the case of inorganic acids, there was protonation of the most volatile wine compounds, which interacted with the fibers.The response of the sensor showed a better linearity for AA than for the inorganic acids. Furthermore, the sensitivity to AA vapors  to a yellow range of 68° to 87° for the hue angle. For doping with SA  in collaboration with FAO’s Rural Infrastructure and Agro-Industries Division, wine quality is measured by chemical analysis, including the volatile acid content , which sor wines . Accordi acidity .−1), increasing to 95° (reference mat subjected to an AA concentration of 10.0 g L−1).The material developed in this study demonstrates potential for use as a wine quality indicator through the color change and color intensity (hue angle) of a nanofibrous mat. The hue angle ranged from 341° (blue color) in the control mat to 82° (reference mat subjected to an AA concentration of 1.2 g LThe incorporation of the BTB pH indicator in PS nanofibers by the SBS technique was performed successfully. In this context, the SBS technique can be considered easy to use for the development of smart nanomaterials with staggered production profiles. The obtained smart nanofibers showed a visual sensitivity to changes in pH in aqueous and vapor media. The incorporation of BTB into the PS fibers did not promote intense modifications to the morphology of the nanofibers but led to the formation of beads that promoted the encapsulation of the dye. The mean PS fiber diameters showed no significant difference, regardless of the BTB concentration used. The migratory potential of the PSNF/BTB-0.2 was considered high for foods with pH < 4.5, but the values obtained in this study were within the limit established by law, especially for beverages with up to 20% alcohol content. Finally, the halochromic potential of the nanofibrous mat was correlated with a rapid chromatic response to red wine vapor, promoting a change in the color of the nanofibers due to the presence of volatile acids in the beverage. These results suggest that the PSNF/BTB-0.2 nanofibrous mats can be used as a nanomaterial sensor for multiple applications dealing with evaluation by direct surface contact or by indirect contact, e.g., through the measurement of volatile acids in the control of wine quality. This advancement opens a door for the use of halophytic nanofiber mats in intelligent packaging as quality sensors for food and beverage products that generate acid products during the spoilage stage.


All effects taken together, bovine tuberculosis (bTB) has a long-term detrimental effect on bovine herds and many wildlife species in South Africa. The disease is not only found in domestic cattle but also in African buffaloes and has to date been diagnosed in 21 wildlife species, including several rare and endangered species, thus having a potentially serious effect on conservation and biodiversity. In cattle, bTB is mostly characterised by sporadic outbreaks, but bovine herds chronically infected with the clinical disease are not uncommon. Presently, the recognised bTB control strategy in South Africa is based on ‘test and slaughter’, using the intradermal tuberculin test, followed by the slaughter of animals that have tested positive. Affected herds are placed under veterinary quarantine with movement restrictions until the outbreak is eradicated; this can take several years or last indefinitely if the outbreak cannot be eradicated. The same measures apply to infected buffalo populations, often with no prospect of ever being eradicated. This strategy is neither practical nor viable in the context of a communal farming system and becomes unethical when dealing with valuable wildlife reservoir hosts. Transmission of bTB between wildlife and cattle has been demonstrated and emphasises the need for an effective, affordable and culturally acceptable control strategy to curb the spread of bTB in South Africa. In countries with similar challenges, vaccination has been used and found to be promising for treating wild and domestic reservoir species and may hence be of value as a complementary tool for bTB control in South Africa. Mycobacterium bovis (M. bovis) with the potential to establish to certain wild animal species. The organism belongs to the so-called Mycobacterium tuberculosis complex, which is characterised by the ability of its members to cause tuberculosis in mammalian species, including humans. Mycobacterium bovis has been circulating within European Mediterranean bovine herds from biblical times  is an economically important and widespread disease in cattle caused by den HSRC .Syncerus caffer) emerged as the maintenance host, with far-reaching implications for multispecies ecosystems because infected buffaloes act as a source of M. bovis to any mammalian species sharing the habitat with them  has shown the potential to act as a maintenance host and most probably responsible for introducing M. bovis into a conservation area , a social species that show predominantly lung and intestinal tuberculous lesions, are considered potential bTB reservoir hosts in ecosystems supporting high population densities   in 1929 (Paine & Martinaglia Potamochoerus larvatus) and baboons (Papio ursinus) in the same reserve in 1998  was an impala that died in 1977, but the diagnosis was never confirmed by culture , suggesting a single introduction into the KNP . It is strongly suspected that amongst the estimated 8000 antelopes translocated into MGR, the M. bovis strain was introduced by another bovid species from KZN. As can be seen from the case above, even under strict biosecurity with regard to cattle and buffalo movements, introduction of M. bovis into an unaffected area by other bovid species is a reality.The Madikwe Game Reserve (MGR), located in the far northern regions of the North West Province, was home to a very valuable disease-freeM. bovis  on commercial ranches  (DAFF M. bovis has been successfully eradicated from the population.The application of control measures for PN) DAFF . For allM. bovis strains into South Africa with infected cattle from Europe during colonial times promoted a high genetic diversity of strains, representing the European (EU)-1 and possibly the EU-2 clonal complexes  cattle farming system, stringent test interpretation (accepting an ‘overkill’ of cattle), financial compensation for cattle slaughtered, adequate government resources guaranteeing completion of the eradication campaign and the absence of an r Amanfu . The sigr Amanfu .In South Africa, buffaloes are highly effective maintenance hosts of bTB, which facilitate the persistence and continued horizontal spread not only to other wildlife species but also to cattle beyond the border of game and veterinary control fences, thus jeopardising bTB control in cattle  of South Africa in 2016 , an alternative bTB test that can be used as an ancillary test at the discretion of the state veterinarian, mitigates the need to confine the buffalo in bomas for 3 days but still necessitates immobilising buffaloes in order to obtain blood samples , community-based animal health care, increased education and involvement of communal cattle farmers with regard to suitable and locally relevant bTB control could be implemented in communal small-scale herds. In addition to all of the above, a vaccination strategy for domestic and wildlife reservoir species could afford the opportunity to significantly reduce the prevalence of diseased animals and limit the onward spread of the disease (Buddle et al. In developed countries with an effective bTB control programme in cattle and a bTB wildlife reservoir host, it has been shown that eradication in livestock depends on concurrent and successful control in the wildlife reservoir (De Lisle et al. M. bovis reservoir species is to induce an immune response that ideally protects the animals against infection. However, incomplete protection, meaning infection not being prevented in the vaccinate, is still highly beneficial if the infection cannot progress to a disease, resulting in significantly reduced disease severity (Palmer & Thacker M. bovis prevalence and can contribute significantly to an integrated bTB control in cattle using lethal and non-lethal approaches (Buddle et al. The aim of vaccinating domestic or wild In wild reservoir hosts, vaccination holds the potential to slow down interspecies transmission to vulnerable wildlife species and also to cattle, which is a major benefit where other control measures are not feasible or not desirable. Bacillus Calmette-Guerin has also been researched in affected wildlife, including European badgers (Chambers et al. M. bovis vaccine have demonstrated protective responses comparable or superior to BCG in a number of wild and domestic animal species, indicating promising prospects for the future use of these vaccines. These inactivated M. bovis vaccines provided good protection in European wild boar (Garrido et al. M. bovis vaccine induced strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination. These cattle were challenged with live BCG vaccine, and although not statistically significant, recovery of BCG after challenge was lowest in the group vaccinated with inactivated M. bovis as compared to BCG and unvaccinated control animals (Van der Heijden et al. On the other hand, the use of a live vaccine in animals destined to enter the human food chain could raise concerns of potentially negative implications for immunocompromised patients (Norouzi et al. In general, more stringent requirements for vaccine use have been postulated for cattle than for wild life. It should be emphasised that this strongly depends on the context in which the vaccine is to be applied. For a vaccine to be considered successful in cattle in epidemiological settings in which conventional control measures are either absent or not implemented because they are deemed impractical, unethical, unaffordable, wasteful or culturally unacceptable, the hallmark of full protection against infection is not justified, similar to the requirements of a wildlife vaccine (Buddle et al. In summary, bTB control in South Africa faces unique challenges within the livestock sector, the wildlife industry and in conservation areas alike, which cannot be successfully addressed using conventional resource-intensive control measures based on culling alone. Vaccination offers a valuable adjunct control tool that has been demonstrated to effectively protect animals from TB disease and can slow down its transmission in multiple species. Prior work conducted in South Africa has established infection models for both cattle and African buffalo, which form a suitable foundation for future vaccine efficacy trials under controlled and field conditions (Buddle et al. It is essential that bTB be well-controlled within South Africa because of the risk it poses to livestock production, commercial game operations, wildlife conservation and human health and livelihoods. However, current approaches to bTB control are not or less effective than required to fulfil the mandate of the Tuberculosis Scheme. The underlying reasons for this inefficacy are manifold and economic, political, technical, social and ethical in nature. To improve the overall bTB control, a new strategy is urgently needed, which takes into account affordability, feasibility and cultural acceptability of the measures to be implemented. Vaccination offers a promising strategy as an integral part of the bTB control in South Africa.


Arabidopsis genome comprises eighty genes encoding BTB  family proteins that are characterized with the BTB domain and that potentially serve as substrate adaptors for cullin-based E3-ligases. In addition to the BTB domain, most BTB proteins also contain various other interaction motifs that probably act as target recognition elements. Here, we report three members of the BTB-A2 subfamily that distinctly only contain the BTB domain, BTB-A2.1, BTB-A2.2, and BTB-A2.3, that negatively regulates abscisic acid (ABA) signaling in Arabidopsis. BTB-A2.1, BTB-A2.2, and BTB-A2.3 encoded cytoplasm- and nucleus-localized proteins and displayed highly overlapping expression patterns in Arabidopsis tissues. Disruption of these three genes, but not single or double mutants, resulted in a decrease in ABA-induced inhibition of seed germination. Further analyses demonstrated the expression levels of these three genes were up-regulated by ABA, and their mutation increased ABA signalling. Importantly, protein-protein interaction assays showed that these three BTB-A2 proteins physically interacted with SnRK2.3. Moreover, biochemical and genetic assays indicated that BTB-A2.1, BTB-A2.2, and BTB-A2.3 decreased the stability of SnRK2.3 and attenuated the SnRK2.3 responsible for the ABA hypersensitive phenotype of seed germination. This report thus reveals that BTB-A2s serve as negative regulators for balancing the intensity of ABA signaling during seed germination.The  Arabidopsis have revealed the core ABA signalling pathway. Without ABA, there is a physical interaction between protein phosphatases 2C (PP2Cs) and sucrose non-fermenting-1-related protein kinases 2 (SnRK2s), which inhibits the phosphorylation activity of SnRK2s and therefore turns ABA signalling off [AtPP2-B11 regulates plants to response to ABA by degrading SnRK2.3 [Seed germination is crucial for next-generation plant growth during the life cycle. Seed germination is frequently a consequence of the competitive interaction between the growth potential of embryonic material and the limited mechanical force of its surrounding tissues, of which the process is elaborately regulated by internal and external signals ,2,3,4. Sling off . In the ling off ,9,10. Thling off ,11. Amonling off ,13. ARK,ling off . Casein ling off . Additio SnRK2.3 . These rArabidopsis, there are about 80 putative BTB proteins split into 10 families (A-J), with two families divided further into two subfamilies (A1/A2 and D1/D2), and functions of several members in this family have been identified [Arabidopsis ethylene overproducer 1 (ETO1), ETO1-like 1 (EOL1), and ETO1-like 2 (EOL2) are involved in the regulation of ethylene synthesis through the recognition and presumed ubiquitination of ACC synthase 5 (ACS5) and related enzymes [Arabidopsis non-expresser of pathogenesis-related genes (NPR1) and NPR3/NPR4 are proposed to be SA  receptors, but play opposite roles in regulating SA-induced plant immunity [Arabidopsis ARIA positively regulates ABA responses possibly through direct interaction with the ABA response transcription factor ABF2 [Protein turnover in most cellular processes requires a tightly controlled coordination between synthesis and degradation, allowing cells to rapidly adapt to various internal and external cues . Proteinentified . For exa enzymes . Arabidoimmunity . Arabidotor ABF2 .Arabidopsis. We employed a reverse genetics method to generate and phenotype the multiple knockout lines of Arabidopsis BTB-A2.1, BTB-A2.2, and BTB-A2.3 to explore their physiological functions. Our genetic data suggested that cytoplasm- and nucleus-localized BTB-A2s functioned redundantly in ABA-induced inhibition of seed germination. We further demonstrated that these three BTB-A2s physically interacted with SnRK2.3 and decreased the stability of SnRK2.3, leading to a decrease in the SnRK2.3 responsible for seed germination. Thus, this study has revealed a previously unrecognized mechanism of maintaining the appropriate ABA signalling level via SnRK2.3 in finely tuned seed germination.Most well-documented BTB proteins belong to the members that contain various other interaction motifs besides the BTB domain. However, the functions of other BTB proteins containing only the BTB domain without other interaction motifs, including the BTB-A2 subfamily, are less known. In this study, we characterized three members of the BTB-A2 subfamily, BTB-A2.1 (AT5G41330), BTB-A2.2 (AT3G09030), and BTB-A2.3 (AT2G24240), in Arabidopsis BTB superfamily, which have additional motifs, the clade A2-type BTB proteins (BTB-A2) only have a BTB domain [Arabidopsis, Drosophila, Homo sapiens, and Cricetulus griseus using these three BTB-A2s members as query sequences. The phylogenetic tree based on the query sequences agreed well with the evolutionary relationship among these species (http://www.cbs.dtu.dk/services/TMHMM-2.0/), BTB-A2.1, BTB-A2.2, and BTB-A2.3 do not have a transmembrane domain  analysis to examine the expression patterns of AtBTB-A2s in 5-week-old Arabidopsis plants and found that the transcripts of BTB-A2.1, BTB-A2.2, and BTB-A2.3 were ubiquitously expressed in all the tissues or organs investigated, with the highest levels in leaves  reporter controlled by the promoter fragment of BTB-A2.1, BTB-A2.2, or BTB-A2.3 and performed histochemical staining of GUS activity on these lines. As shown in BTB-A2.1, BTB-A2.2, and BTB-A2.3 were highly similar, suggesting that they have a possible overlap of functions.To identify the plant tissues where BTB-A2s may function in n leaves C. In addArabidopsis plants, we screened the putative transfer deoxyribonucleic acid (T-DNA) insertion mutants of the BTB-A2.1, BTB-A2.2, or BTB-A2.3 gene obtained from the Arabidopsis Biological Resource Center (ABRC) and confirmed the positions of gene knockout by PCR analysis and DNA sequencing  plants during the life cycle, including vegetative and reproductive periods , a putative precursor of SA, or p-hydroxybenzoic acid (pHBA), an isomer of SA [btb-a2.1/2/3 plants and found these two plants displayed a similar root growth phenotype under these treatments -related phenotypes in WT and er of SA , to treaeatments . Becausend light . We founnd light . These rcis-elements in the promoter region of BTB-A2.1, BTB-A2.2, or BTB-A2.3 using PlantCARE analysis  in WT and the btb-a2.1/2/3 triple mutant were also analyzed. As expected, these genes were induced by exogenous ABA in WT, in agreement with previous studies [btb-a2.1/2/3 triple mutant were markedly elevated compared with those in WT plants  assay  assay . These rArabidopsis BTB-A2.1, BTB-A2.2, and BTB-A2.3 have the core structure of the BTB domain and protein–protein interaction domain, which may act as the substrate adaptors for CUL3-based E3-ligases [Arabidopsis BTB-A2.1, BTB-A2.2, and BTB-A2.3 with SnRK2.3 in plant cells, we speculated whether BTB-A2.1, BTB-A2.2, and BTB-A2.3 are involved in ABA signalling by mediating the stability of SnRK2.3. We constructed 35S::SnRK2.3-Flag transgenic plants in WT and the triple mutant btb-a2.1/2/3 background, respectively. Seven-day-old transgenic seedlings were treated with 50 µM cycloheximide  for the specified point-in-time, and then proteins were extracted. The SnRK2.3 content was detected with flag antibody. Western blotting showed that the SnRK2.3 protein level decreased more slowly in the btb-a2.1/2/3 mutant than that in WT at the corresponding time (35S::BTB-A2.1 overexpression (BTB-A2.1-OE), 35S::BTB-A2.2 overexpression (BTB-A2.2-OE), and 35S::BTB-A2.3 overexpression (BTB-A2.3-OE) transgenic lines. Those BTB-A2s-OE transgenic lines that displayed the highest BTB-A2s expression levels were selected for further analysis  is the basis of the diverse aspects of eukaryotic cell regulation by precise modulation of the stability of short-lived and abnormal intracellular proteins and also by the modulation of phytohormone signalling by affecting protein activity, localization, assembly, and interaction ability . The ubi complex ,38,39. IArabidopsis BTB-A2.1, BTB-A2.2, and BTB-A2.3 localized in both the cytoplasm and nucleus  seedlings were used in the research. The T-DNA insertion mutants AT5G41330 (SALK_114523C), AT3G09030 (SALK_101331), and AT2G24240 (CS825825) were obtained from the Arabidopsis Biological Resource Center. The positions of T-DNA insertion sites are shown in Arabidopsis seeds were sterilized with 75% ethanol for 5 min, washed three times with sterilized water and then sown on 1/2 MS supplement with 1% (w/v) sucrose and 0.8% (w/v) phytoblend agar . The pH was 5.8. The seeds were stratified at 4 °C for two days and were then placed in a growth chamber  at 22 °C. For soil culture, 10-day-old seedlings on 1/2 MS were transferred to nutrient-rich soil  and then grown in a greenhouse with a long-day cycle  at 22 °C.For on-plate growth assays, the BTB-A2.1, BTB-A2.2, and BTB-A2.3 overexpression transgenic plants, the full-length coding sequence (CDS) of BTB-A2.1, BTB-A2.2, BTB-A2.3 was amplified from the cDNA of WT and then introduced into the binary vector pCAMBIA-3301 under the control of the CaMV35S promoter. To generate the proBTB-A2.1::BTB-A2.1-GFP construct, we fused the GFP and NOS terminator sequence with the 3609-bp genomic DNA (stop codon was deleted) of BTB-A2.1, including the 2232-bp promotor sequence and the 1377-bp coding sequence, and then inserted this recombinant DNA into the binary vector pCAMBIA-1300. The pBTB-A2.2::BTB-A2.2-GFP, pBTB-A2.3::BTB-A2.3-GFP constructs were obtained using the same method mentioned above. To generate the pBTB-A2.1::GUS construct, we amplified the promoter of BTB-A2.1 from WT genomic DNA and introduced this into the modified binary vector pCAMBIA-1300 harboring the GUS reporter gene. pBTB-A2.2::GUS, pBTB-A2.3::GUS constructs were obtained using the same method mentioned above. To generate SnRK2.3-OE transgenic plants, the full-length CDS of SnRK2.3 was amplified from the cDNA of WT and then cloned into the binary vector pCAMBIA-1302 under the control of the CaMV35S promoter. Those constructs were introduced into the Agrobacterium tumefaciens GV3101 strain to transform into Arabidopsis plants using the floral dipping approach [To generate approach . The priACTIN2 (AT3G18780) was used as the internal standard in both RT-PCR and qPCR analyses. All individual reactions were performed in triplicate. All gene-specific primers used are listed in Total RNA was extracted from plant samples using TRIzol reagent  according to the manufacturer’s instruction. First-strand cDNA synthesis was performed using M-MLV Reverse Transcriptase  according to the manufacturer’s instruction. The RT-PCR analysis of gene expression using the cDNA was followed by 27 cycles of PCR. qPCR was performed using an SYBR Green I Master kit  according to the manufacturer’s instructions on a CFX Connect Real-Time System . BTB-A2.1 was inserted into the pEZS-NL-GFP, which generated a C-terminal fusion with the GFP gene controlled by the CaMV35S promoter. The stop codon was deleted. 35S::BTB-A2.2-GFP and 35S::BTB-A2.3-GFP constructs were obtained using the same method mentioned above. The primers used are listed in For the subcellular localization analysis, the full-length CDS of Arabidopsis protoplasts were conducted according to a published protocol [Isolation and transient expression in protocol . ImagingThe GUS staining was performed according to a published protocol . BrieflyBTB-A2.1, BTB-A2.2, BTB-A2.3 or SnRK2s  was inserted into pGADT7 (AD) and pGBKT7 (BD). The fusion AD and BD constructs were co-transformed into yeast strain AH109 using the lithium acetate transformation method [−1, 10−2, and 10−3) at 30 °C for 3–5 days. The primers used are listed in Full-length CDS of n method . The traBTB-A2.1, BTB-A2.2, or BTB-A2.3 was fused in-frame to the C-terminus of YFP to form BTB-A2.1-cYFP, BTB-A2.2-cYFP, and BTB-A2.3-cYFP. The full-length CDS of SnRK2.3 and SnRK2.6 was cloned in-frame into the N-terminus of YFP to generate SnRK2.3-nYFP and SnRK2.6-nYFP, respectively. All of the constructs were transformed into A. tumefaciens strain GV3101 and then infiltrated into Nicotiana benthamiana leaves following the method reported by Hu et al. [Full-length CDS of u et al. . InfecteArabidopsis seedlings were treated with CHX at the indicated times. Seedlings were harvested and frozen in liquid nitrogen for protein extraction, which was performed as described [Seven-day-old escribed . For immt-test (* p < 0.05 and ** p < 0.01) or one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (p < 0.05).For all experiments, data were analyzed using Excel and Origin 8. Mean values ± SD of at least three replicates are presented. Data were subjected to statistical analyses using Student’s


Drew  Impulse control disorders in Parkinson’s disease are common neuropsychiatric complications associated with dopamine replacement therapy. Some patients treated with dopamine agonists develop pathological behaviours, such as gambling, compulsive eating, shopping, or disinhibited sexual behaviours, which can have a severe impact on their lives and that of their families. In this study we investigated whether hypersensitivity to reward might contribute to these pathological behaviours and how this is influenced by dopaminergic medication. We asked participants to shift their gaze to a visual target as quickly as possible, in order to obtain reward. Critically, the reward incentive on offer varied over trials. Motivational effects were indexed by pupillometry and saccadic velocity, and patients were tested ON and OFF dopaminergic medication, allowing us to measure the effect of dopaminergic medication changes on reward sensitivity. Twenty-three Parkinson’s disease patients with a history of impulse control disorders were compared to 26 patients without such behaviours, and 31 elderly healthy controls. Intriguingly, behavioural apathy was reported alongside impulsivity in the majority of patients with impulse control disorders. Individuals with impulse control disorders also exhibited heightened sensitivity to exogenous monetary rewards cues both ON and OFF  dopamine medication, as indexed by pupillary dilation in anticipation of reward. Being OFF dopaminergic medication overnight did not modulate pupillary reward sensitivity in impulse control disorder patients, whereas in control patients reward sensitivity was significantly reduced when OFF dopamine. These effects were independent of cognitive impairment or total levodopa equivalent dose. Although dopamine agonist dose did modulate pupillary responses to reward, the pattern of results was replicated even when patients with impulse control disorders on dopamine agonists were excluded from the analysis. The findings suggest that hypersensitivity to rewards might be a contributing factor to the development of impulse control disorders in Parkinson’s disease. However, there was no difference in reward sensitivity between patient groups when ON dopamine medication, suggesting that impulse control disorders may not emerge simply because of a direct effect of dopaminergic drug level on reward sensitivity. The pupillary reward sensitivity measure described here provides a means to differentiate, using a physiological measure, Parkinson’s disease patients with impulse control disorder from those who do not experience such symptoms. Moreover, follow-up of control patients indicated that increased pupillary modulation by reward can be predictive of the risk of future emergence of impulse control disorders and may thereby provide the potential for early identification of patients who are more likely to develop these symptoms. Idiopathic Parkinson’s disease is now known to be associated with a wide range of non-motor symptoms including potential impairment in cognition, mood and motivation . ImpulseThe more severe forms of behaviour, meeting formal diagnostic criteria for a ‘disorder’, are reported at a prevalence of ∼15–25% of the Parkinson’s disease population . The difParkinson’s disease itself is not considered to carry increased risk for development of ICBs  were recruited to take part in this study . Two patients were not included in the analysis because eye tracking data could not be obtained due to technical difficulties; therefore, the data from 23 PD+ICD cases are presented here. Twenty-six Parkinson’s disease cases  without impulse-control behaviours (PD-no-ICD) were included in the study. At the time of testing, none of these patients reported a history of or current diagnosis of ICD. Thirty-one elderly healthy controls were also tested. The performance on the task of PD-no-ICD and healthy controls was previously reported in Despite often co-occurring in the same patient, it has been proposed that ICBs such as dopamine dysregulation syndrome, and to a lesser extent hobbyism and punding, may have different underlying physiological substrates from ICDs (All 23 PD+ICD patients tested were established on dopaminergic medication (levodopa and/or dopamine agonists). Eleven PD+ICD patients were on levodopa only, none were on only dopamine agonists and 12 were on a combination of both . PatientIn the PD-no-ICD group, 15 patients were on levodopa only, and 11 were on a combination of levodopa and dopamine agonists. We found no evidence that any of the 15 PD-no-ICD patients only currently on levodopa had previously taken dopamine agonists. It is, of course, possible that this could reflect reverse causation in the recruitment of patients, that the decision to start an agonist might have been avoided in subjects with greater impulsivity or patients with a high risk of developing ICDs. However, we have no data on this aspect of clinical decision-making for these individuals.PD+ICD cases were administered the short form of the QUIP known as the QUIP-S. This is a self-report questionnaire, consisting of two parts, QUIP-anytime and QUIP-current . The BISApathy was assessed with the Lille Apathy Rating Scale (LARS), a semi-structured clinical interview which has previously been validated in Parkinson’s disease  while participants made eye movements toward targets presented on a computer screen. They were instructed to make quick, direct eye movements and were told that the faster that they looked at peripheral targets, the greater a proportion of the reward on offer they would receive . In eachIn each trial, participants received a proportion of the maximum reward on offer dependent on their saccadic performance. The absolute amount of reward they received varied with reaction time, but importantly this amount was dynamically adjusted using an ‘adaptive’ exponential fall-off based on average reaction time of the 20 previous trials . This alIn all trials a saccade to the peripheral target was required in order to move to the next trial, including in the 33% of trials in which there was no reward stimulus and no reward on offer (the 0p condition).All participants performed five experimental blocks consisting of 54 trials and were given a 5-min break between the third and fourth blocks. Therefore, they each performed 270 trials in total, 90 for each reward condition. Disc luminance and ambient room lighting was consistent across all trials to avoid differentially affecting pupil dilation. If a reward of 10p or greater was earned, an audible bell sound was played, and for rewards of 30p or greater the sound of a cash register was played concurrently with the appearance of the visual feedback. For earned rewards of less than 10p no sound was played but for 0p a low frequency buzzing sound was played. Behavioural indicators of task performance included saccadic speed (mean peak velocity) and saccadic variability [standard deviation (SD) of saccadic amplitude].Participants sat 60 cm from a 21′′ CRT computer screen  in a dimly lit room. Visual stimuli were presented using MATLAB (The MathWorks) and Psychophysics Toolbox on a Microsoft Windows PC. The frame mounted infrared eye tracker monitored left eye position at a sampling rate of 1 kHz and a 9-point calibration was performed. The Eyelink computer measured eye movements online and fed back into the presentation computer to provide immediate feedback.Analysis of the eye tracking data was performed using standard Eyelink criteria, with a velocity and acceleration threshold for saccade initiation. Saccades were defined as eye movements >2° and were considered to be complete when they landed within a 5° radius from the target centre. Reaction times were calculated as the time when the target first appeared to the time of saccade completion and peak saccadic velocity was calculated as the maximum speed of the eye position trace in windows of 3 ms, measured for the first saccade that landed outside the central fixation disc. Pupil trajectories which were lost due to blinking were interpolated up to a maximum of 500 ms, trials with larger gaps were discarded. Pupil dilation was measured as the proportional change from the average baseline pupil size. For each trial, recordings were time locked to the onset of the reward cue and normalized by subtracting the baseline pupil size at 0 ms. A moving average smoothing window of 10 ms, then 100 ms was applied.P-values were calculated for every time point after 600 ms. Where appropriate, statistics were reported with Greenhouse-Geisser corrections, to account for non-sphericity in the data. If significant interactions were observed, corrected pairwise comparisons were made using Bonferroni post hoc tests. Significance was taken as P-values of < 0.05. For non-parametric questionnaire data, Spearman Rank correlations were performed, and Pearson correlations were utilized for parametric testing of behavioural eye tracking data. Bonferroni corrections for multiple correlations were performed. Statistics were performed using MATLAB, SPSS and JASP.Analysis of pupillary and saccadic data was performed using split-level ANOVA, with within-subjects factors of Drug  and Reward , and between-subjects factor of Clinical Group . For each saccadic and pupillary variable a new variable reflecting reward sensitivity was calculated as the difference in mean proportional pupil change between the 50p and 0p reward conditions (50p − 0p) and further analyses were performed using repeated measures ANOVA, with a within-subjects factor of Drug  and a between-subjects factor of Clinical Group . Significance in the pupil trace figures was calculated using a permutation analysis whereby https://www.opdc.ox.ac.uk/external-collaborations for further information.Applications for deidentified demographic data can be made to the Oxford Parkinson’s Disease Centre. See There were no significant differences between the PD+ICD and PD-no-ICD groups in age, cognitive impairment (MoCA), depression (BDI) or anhedonia (SHAPS/TEPS), or anxiety  . Howevert(31.073) = 3.057, P = 0.005] and in the subscales; UPDRS Part 1 , Part 2 , Part 4 , and Hoehn and Yahr stage , reflecting overall greater Parkinson’s disease symptom severity in PD+ICD.Furthermore, the two groups differed in UPDRS Total , and emotional apathy with PD+ICD exhibiting greater levels of emotional apathy than PD-no-ICD .There was significant comorbidity of apathy and impulsivity. Of PD+ICD patients, 78.3% were either borderline (43.5%) or clinically (34.8%) apathetic. This is a higher proportion than in the PD-no-ICD group in which 46.2% were either borderline (11.5%) or clinically (34.6%) apathetic as measured by the LARS clinical interview. There were no significant differences between PD+ICD and PD-no-ICD in LARS total, or in the motivational domain of intellectual curiosity (cognitive apathy). Furthermore, the groups did not differ significantly in the self-awareness component of the LARS. However, they did differ significantly in two of the three motivational domains: action initiation  with the PD-no-ICD group scoring significantly better (higher levels of action initiation) than PD+ICD . Across both groups, as reported previously in Parkinson’s disease  = 29.694, P < 0.001]. There was no significant difference in this dopaminergic effect between the groups, i.e. baseline pupil size ON dopamine minus OFF dopamine . Note that our interest in this study is in the subsequent impact of the reward incentives on changes from baseline–pupil reward sensitivity, discussed below. The key points from the baseline pupil size analysis show that PD+ICD patients had larger pupils, so if anything, they have less potential dynamic range for further dilation to reward, and all patients have bigger pupils ON dopamine, again meaning that there is less potential dynamic range for more dilation.We first examined baseline pupil size to ensure it could not account for the reported group differences on reward sensitivity. Baseline pupil size for each patient was measured as the mean pupil diameter (in arbitrary units) across all experimental reward conditions  at the beginning (0 ms) of each trial, at the time of onset of the auditory reward cue. PD+ICD patients had greater mean pupil sizes than PD-no-ICD   = 5.692, P = 0.005] reflecting differences in the dopaminergic effect on pupil reward sensitivity in the two groups. Furthermore, there was a main between-subjects effect of group (PD+ICD versus PD-no-ICD) revealing overall smaller pupil responses to reward in PD+ICD , as well as a main effect of drug condition with greater overall pupil response when OFF dopamine in both groups . There were no two-way interactions.In both PD+ICD and PD-no-ICD, pupils dilated more in response to increasing monetary reward  . In addiThese results indicate that overall there was less pupillary dilation while ON dopamine. However, this does not reflect reward insensitivity. Rather, dopamine has a mydriatic effect on the pupil, increasing baseline pupil size and hence allowing less scope for further dilatory response to reward. Our primary interest here is relative reward sensitivity. Despite reduced overall dynamic pupil change while ON dopamine, reward sensitivity (i.e. greater pupil dilation for greater reward on offer) effects were observed in both groups.F = 5.336, P = 0.008; F = 7.044, P = 0.002], whereas dopamine reduced it . The interaction in the PD-no-ICD group is indicative of a steeper reward sensitivity slope ON medication compared to OFF  repeated measures ANOVA. In the PD-no-ICD group, being ON dopamine led to a significant increase in reward-related pupil dilation  whereas reward increased it . Bonferroni post hoc pairwise comparisons between the three reward levels  demonstrated significant differences in pupillary reward response between each level (P < 0.01). In the PD+ICD group, there was also no significant difference between the sensitivity slopes ON and OFF dopamine  = 8.643, P = 0.005]. For PD+ICD cases, there was no significant difference compared to healthy controls, either in the ON or OFF state. Analysis of the difference in pupillary reward sensitivity ON versus OFF dopaminergic medication (ON − OFF) demonstrated a significant difference between PD+ICD and PD-no-ICD groups . These findings highlight that, quite different to our initial hypothesis, PD+ICD cases do not in fact show enhanced reward sensitivity ON dopaminergic medication, but rather show enhanced sensitivity to reward both ON and OFF, with no significant difference between these states, unlike PD-no-ICD patients who show greater sensitivity ON as previously reported for patients with Parkinson’s disease , depression (BDI), anhedonia (SHAPS/TEPS), or anxiety (DASS) . FurtherTo investigate the role of potential confounds more closely, we assessed the effects of: age, disease severity, disease duration, currently active versus previously active ICD, current dopamine agonist therapy (with levodopa) versus levodopa therapy alone, and cholinergic medicationsrS = −0.359, P = 0.011), the motor symptoms section; there was a trend towards correlation with UPDRS Part 1 which assesses non-motor symptoms . Total UPDRS also trended towards significance . Within each group, there were no significant correlations; this might reflect sample size. Overall, patients with more pronounced motor symptoms (UPDRS Part 2), therefore had weaker effects of dopamine on reward sensitivity, i.e. a smaller increase in reward sensitivity when ON compared to when OFF dopaminergic medication.There were no significant correlations between age and pupil reward sensitivity ON or OFF dopamine, or between age and the effect of dopamine on reward sensitivity (RS ON −OFF). PD+ICD cases had higher symptom severity scores, as measured by the UPDRS . We therWe next asked whether this correlation in drug effect was driven by being ON or by being OFF drug. However, no significant correlations were found between UPDRS total or subscales and pupillary reward sensitivity either ON or OFF dopamine in the PD+ICD group or the whole Parkinson’s disease population ON or OFF dopamine. Thus, it was specifically the difference in response to reward ON versus OFF dopamine that was negatively correlated with motor symptom severity. There was no significant effect of disease duration (from time of diagnosis). However, patients with longer prodrome periods (difference in time between symptom onset and disease diagnosis) were more likely to have larger effects of dopamine on pupillary reward sensitivity .post hoc subgroup analysis to assess whether there were differences in pupil reward sensitivity in PD+ICD patients with currently active (n = 15) versus previous ICD (n = 8) symptoms and found no significant differences between these groups (n = 12) with those who only take levodopa (n = 11). We found no significant differences between these subgroups in terms of proportional pupil change reward sensitivity ON dopamine. However, PD+ICD patients currently on agonists had significantly greater pupil response to reward OFF dopamine , mean of ON and OFF dopamine  but crucially not in the effect of dopamine on reward sensitivity (RS ON-OFF) [P = 0.380]  were on levodopa and were compared to the PD-no-ICD cases (n = 26). There were significant main effects of reward , drug (ON versus OFF)  and group  as well as a significant 3-way interaction between reward × drug × group . Thus, even when excluding all PD+ICD patients who were currently taking dopamine agonists at the time of testing the main findings reported in the text are preserved, despite a significantly smaller sample size in PD+ICD cases who were not dopamine agonists, but taking only levodopa.To assess whether the overall PD+ICD result of greater reward sensitivity in the OFF condition compared to PD-no-ICD cases could all be attributed to the PD+ICD cases on dopamine agonists, we performed another rS = 0.322, P = 0.024) and the mean for ON and OFF . Thus, increased DA-LEDD predicts increased proportional pupil change OFF medication, and mean overall proportional pupil change, but not ON medication or crucially the difference between ON and OFF.We also examined the effect of LEDD and DA-LEDD (dopamine agonist LEDD). There were no significant correlations between LEDD and pupil reward sensitivity ON, OFF, overall mean or ON-OFF. However, there were significant correlations between DA-LEDD and pupil reward sensitivity OFF  was a significant factor affecting their reward sensitivity. We obtained the dopamine agonist withdrawal dates for five cases. For these patients the average time of withdrawal before testing on our oculomotor task was 5.4 years (range: 2.5–10 years). While it is possible that ongoing subclinical effects were present, it is quite unlikely that this was a significant factor as DAWS usually does not affect patients for this long. Approximately half of DAWS cases resolve themselves within days or weeks but in more serious cases it can last months or years .n = 1, donepezil n = 1). In the PD-no-ICD group, only one patient was currently taking cholinergic medication (trihexyphenidyl). These medications are known to have direct mydriatic effects on the pupil. We reanalysed the data after excluding all patients who were taking cholinergic medication and the overall findings were not affected , and those who did not . Pupillary reward sensitivity ON dopamine was greater in the QUIP > 0 group than the QUIP = 0 group , demonstrating that exogenous monetary reward invigorated saccadic responses across both groups. Peak velocities were actually faster OFF drug , as in previous studies  = 4.669, P = 0.012] with greater reward sensitivity ON dopamine. There was a trend towards greater invigoration by reward in PD+ICD compared to PD-no-ICD, but this was not significant  and there was no significant three-way interaction [drug × reward × group (P = 0.675)]. There were no significant differences between the two groups in mean peak velocity or interaction between drug condition and group  = 18.599, P < 0.001]. This occurred more when ON drug . Thus, the observed effect of reward cues on peak velocity is not attributable to the increased amplitudes. This analysis revealed no significant main effect of drug, of group or the interaction between reward and group, demonstrating that PD+ICD do not have greater reward sensitivity of velocity residuals or overall faster saccades than PD-no-ICD. There were no other significant two- or three-way interactions, but we note that the overall pattern of results  = 3.795, P = 0.026], but no other main effects or interactions were found (Variability (SD) of saccadic amplitude can be understood as a measure of saccadic precision. Lower amplitude variability reflects greater accuracy of saccadic amplitude while, conversely, greater amplitude variability is indicative of greater saccadic imprecision. According to the speed-accuracy trade-off, faster eye movements should result in larger error; however, reward has been shown to violate this trade-off  = 5.293, P = 0.026], and reduced variability of reaction time  compared to PD-no-ICD.In this simple prosaccade task, reward magnitude had no effect on saccadic reaction time, in either group. Furthermore, there was no observed difference between the two groups in terms of reaction time reward sensitivity, and no effect of dopamine on reaction times. PD+ICD did, however, have faster overall reaction time .β = −0.456, P < 0.001), BIS , disease duration  and symptom duration  scores were moderately predictive of the effect of dopamine on pupil reward sensitivity . Thus, increased fun-seeking, behavioural inhibition, disease duration and decreased symptom duration was predictive of a decreased effect of dopamine on pupillary reward sensitivity. This is in line with our finding that the PD+ICD group had on average more severe Parkinson’s disease symptoms and greater behavioural impulsivity, as well as reduced effect of dopamine on pupil reward sensitivity. There was a trend towards a negative correlation indicating that with increasing overall Parkinson’s disease symptom severity , patients exhibit decreasing effect of dopamine on reward sensitivity .A second linear regression analysis was performed with the same predictor variables, this time with pupil reward sensitivity ON minus OFF as the dependent variable. In the whole Parkinson’s disease population, we found that BAS Fun-seeking  dopamine medication, as indexed by pupillary dilatation in response to reward cues. In contrast, PD-no-ICD patients show greater reward sensitivity ON medication compared to OFF . FurtherThe pupillary reward sensitivity findings were independent of total LEDD at the group level and did not significantly correlate with it across patients. However, there were some effects that suggest a potential impact of dopamine agonists. Across the patient groups, DA-LEDD was significantly correlated with pupil reward sensitivity when OFF dopaminergic medication and with respect to the mean of ON and OFF. But increased DA-LEDD did not predict increased proportional pupil change ON medication or crucially the difference between ON and OFF.cf.PD+ICD patients on agonists as a group compared to those on levodopa only had significantly greater pupil response to reward when OFF or with respect to the mean of ON and OFF dopaminergic medication. However, there was no significant difference between the groups when ON, nor crucially with respect to the effect of dopaminergic medication on reward sensitivity ON-OFF. Furthermore, when PD+ICD patients who were currently taking dopamine agonists were excluded and only those patients on only levodopa were compared to the PD-no-ICD cases, the main findings remain unchanged and significant cf.. Taken tThe results of many different studies have pointed to the possibility that ICDs in Parkinson’s disease might develop in response to dopamine agonist treatment interacting with underlying Parkinson’s disease pathophysiology and possibly personality traits .The findings reported here also suggest that the impulsive behaviours observed in PD+ICD patients might not be simply due to a direct effect of dopaminergic drug level on their sensitivity to reward. There was no difference in reward sensitivity between patient groups when ON dopamine medication, suggesting that impulse control disorders may not emerge simply because of a direct effect of dopaminergic drug level on reward sensitivity. It has previously been demonstrated that insensitivity to reward is an important factor in apathy in Parkinson’s disease and dopaminergic medication can improve motivation . These findings are in keeping with the proposal that ICD is likely to be a manifestation of an underlying phenotype conferring propensity to develop impulsive behaviours, which these patients share.b; Proposed risk factors for the development of ICBs and ICDs include younger age and younger age at Parkinson’s disease onset and longer disease duration  in order to obtain the reward offered. This suggests that these patients may be under-motivated when there are no immediate extrinsic reward cues, contributing to behavioural apathy, but over-motivated when responding to the presentation of, for instance, extrinsic monetary reward. Another possible interpretation of the counter-intuitive comorbidity of apathy and impulsivity is that these patients could experience heightened sensitivity for immediate pleasures, or associated reward cues while having difficulty generating or pursuing long term goals. Finally, it is worth noting that neurotransmitters other than dopamine, such as serotonin and noradrenaline, are known to be involved in arousal, incentive salience and reward processing . Barber There were several limitations of this study. First, PD+ICD patients were recruited from a single tertiary centre from (unselected) referrals from several hospitals, so the patient groups might not be representative of typical cases. Although we report the data from 49 Parkinson’s disease cases ON and OFF medications, the sample sizes are relatively small. The PD+ICD group contained individuals with current and previous ICDs and was therefore potentially heterogeneous and there were baseline factors that might also have been potential confounding factors, although we have tried to control for all these issues in the analyses performed herein.This study demonstrates that PD+ICD patients have heightened sensitivity to exogenous monetary reward cues both ON and OFF  dopamine medication, whereas PD-no-ICD patients show increased reward sensitivity ON medication compared to OFF. Although the effects are not accounted for by total LEDD, there is an impact of DA-LEDD on pupil reward sensitivity, but this cannot account for the entire pattern of results. Moreover, the difference between PD+ICD and PD-no-ICD groups remains significant even when PD+ICD cases on dopamine agonists are excluded. The findings support the view that ICDs emerge in Parkinson’s disease in response to dopamine replacement therapy and that hypersensitivity to rewards in some susceptible individuals might contribute to impulsive behaviours in Parkinson’s disease. However, the findings argue against a simple relationship between dopamine level and reward sensitivity. Many PD+ICD patients were also found to experience behavioural apathy and impulsivity comorbidly, suggesting that aberrant reward sensitivity is just one component of a dysfunctional system which may incorporate functional changes in other neurotransmitter systems. The simple oculomotor paradigm employed here also provides the potential for novel clinical measurement of impulsivity and could be used to identify patients who are at greater risk to develop ICDs in the future.This work was supported by a Wellcome Trust Principal Research Fellowship to MH, the National Institute for Health Research Oxford Biomedical Research Centre, a Wellcome Trust Clinical Research Training Fellowship to KM and a Medical Research Council Clinician Scientist Fellowship MR/P00878X to SM.The authors report no competing interests.awaa198_Supplementary_DataClick here for additional data file.


The cardiorenal syndrome relates to the detrimental interplay between the vascular system and the kidney. The uremic milieu induced by reduced kidney function alters the phenotype of vascular smooth muscle cells (VSMC) and promotes vascular calcification, a condition which is strongly linked to cardiovascular morbidity and mortality. Biological mechanisms involved include generation of reactive oxygen species, inflammation and accelerated senescence. A better understanding of the vasotoxic effects of uremic retention molecules may reveal novel avenues to reduce vascular calcification in CKD. The present review aims to present a state of the art on the role of uremic toxins in pathogenesis of vascular calcification. Evidence, so far, is fragmentary and limited with only a few uremic toxins being investigated, often by a single group of investigators. Experimental heterogeneity furthermore hampers comparison. There is a clear need for a concerted action harmonizing and standardizing experimental protocols and combining efforts of basic and clinical researchers to solve the complex puzzle of uremic vascular calcification. Chronic kidney disease (CKD) is recognized as a major non-communicable disease of growing epidemic dimension worldwide. The prevalence of CKD has increased by 19.6% between 2005 and 2015 , the majVC as consequence of EVA can occur at two distinct sites: in the intimal layer of vessels where it is associated with atherosclerosis and in the medial layer, where it is also referred to as Mönckeberg sclerosis. Medial calcification is linked to non-traditional risk factors and is a common feature in CKD, which often co-exist with intimal calcification ,16. ArteUraemia (in Greek) literally means “urine in the blood”. A key future of the uraemic syndrome is the accumulation of URMs. URMs comprise an extremely heterogenous group of molecules of different origins, molecular weight (MW), biological functions and physico-chemical properties ,27. TheiThe present literature review aims to update current evidence of the role of URMs in VC . A totalMM are predominantly proteins and peptides generated endogenously as a response to uraemia, such as cytokines and peptide hormones. Dialysis has limited efficacy in controlling MM, partly related to size restriction of the pores of dialyzers . Of the PBURM are a heterogenous group of solutes, characterized by reversible protein binding capacity. PBURM are considered a major threat to ESRD patients, partly because of their limited removal by conventional dialysis . Of the The group of LMWS comprises compounds with molecular weight <500 kDa and minimal protein binding. Reduction rates of LMWS during dialysis are overall high, but show substantial variability . Twelve Overall, information on the impact of uremic toxins on the process of vascular calcification, in all its aspects, is fragmentary  and by l2+ or Pi. Also, for Ca2+ or Pi, concentration, donor , exposure time and the use of foetal bovine serum (FBS) are subject to differences. A proposal for standardization has recently been put forward, however consensus has not been reached yet [Experimental heterogeneity hampers comparison of data. Primary human, bovine, mouse and rat VSMCs as well as cell lines have been used by different investigators. Interspecies differences may account at least partly for inconsistent findings. VSMC derived from different topographic locations of the vascular tree may differ in their physiology and pathophysiology . In addiched yet . Additioched yet . This reched yet ,288. In As discussed earlier, URM may affect the process of calcification also by indirectly targeting VSMC. For instance, urea and thiocyanate serve as substrates for carbamylation of LDL and proteins ,290, an Literature also inheres a considerable inconsistency in effects of URM on VC between experimental and clinical studies. Biomarker assays only reflect a snapshot in time and neglect the slow progressive nature of CKD  and its The conflicting results obtained in clinical and in vitro studies preclude strong conclusions. Furthermore, consistency of the findings is often limited. Indeed, 15 out of 26 URM have been tested only once in vitro and thus lack independent confirmation. Additionally, we found inconsistencies between clinical studies investigating the same URM. Both case-mix and variable residual confounding may account for these inconsistencies. Furthermore, clinical studies can only indicate correlation and not prove causation. In this context also correction for confounding factors might be challenging giving the multifaceted nature of CKD and the huge body of yet explored influences of the URM. Moreover, the Bradford Hill criteria  have notUremic serum and Pi induces phenotypic modulation and calcification of VSMC differently, thereby underscoring the importance of the uremic toxins as contributors to VC . HoweverThese findings call for a more standardized and systematic approach in order to ensure relevance of results for the therapeutic setting and to facilitate comparability between studies.Although there is currently no approved therapy to stop, attenuate or regress VC there are several appealing approaches ,295. TheThe composition of the uremic serum differs vastly among CDK patients. Thus, although CKD patients suffer from VC, the underlying cause for VC development may differ. In order to progress towards more personalized CKD and CRS treatment strategies a better understanding of the individual uremic toxin profile is needed. This might pave the way for the early identification of patients with a higher risk for VC development and accelerated progression. Thus far, comprehensive URM profiles of individual patients are not available and information stem from measurements of a limited number of URM. Moreover, since the list of URM is constantly updated, it is difficult to define all molecules relevant in the context of VC . A comprIn this review, we included literature investigating URM and VSMC calcification in vitro, in vivo and on a clinical level. A clear definition of URM being detrimental, harmless or beneficial with respect to vascular calcification is currently lacking. Furthermore, we identify many gaps in URM research related to VSMC. A large fraction of URM has not been investigated with respect to their effects on VSMC calcification. However, some are linked to processes indirectly associated with VSMC calcification, such as ROS and inflammation. We also include potential novel avenues of VSMC driven calcification, such as extracellular vesicles, senescence and the influence of the microbiome. Effects of URM are often reported once in publications, and experimental conditions often lack an adequate degree of standardization and are not designed in relation to CRS. The composition of uremic serum is divers and may differ vastly between patients. Moreover, the influence of many uremic retention molecules on VSMC and VC, remain largely obscure and the underlying effects poorly understood. Better understanding of the effects of URM on the vasculature could be the first step towards reducing VC and a personalized CKD treatment strategy. This underscores the need for deep patient serum profiling to understand the underlying cause and consequence relationship between URM and clinical manifestations. In conclusion, our narrative overview puts forward the hypothesis to test effects of URM on VSMC in relation to VC more systematically and advocates for more CRS centred research. The European uremic toxin work group has created an extensive database  containi


In this review, we describe recent relevant advances in the fabrication of polymeric nanofibers to address challenges in conventional approaches such as electrospinning, namely low throughput and productivity with low size uniformity, assembly with a regulated structure and even architecture, and location with desired alignments and orientations. The efforts discussed have mainly been devoted to realize novel apparatus designed to resolve individual issues that have arisen, i.e., eliminating ejection tips of spinnerets in a simple electrospinning system by effective control of an applied electric field and by using mechanical force, introducing a uniquely designed spinning apparatus including a solution ejection system and a collection system, and employing particular processes using a ferroelectric material and reactive precursors for atomic layer deposition. The impact of these advances to ultimately attain a fabrication technique to solve all the issues simultaneously is highlighted with regard to manufacturing high-quality nanofibers with high- throughput and eventually, practically implementing the nanofibers in cutting-edge applications on an industrial scale. Nanofibers belong to a group of most interesting and promising materials due to their unique physicochemical properties, i.e., extraordinary porosity with interconnectivity between pores in mats, mechanical flexibility and strength, high surface area, and high applicability to fabricate composites with other materials ,2,3. TheIn general, electrospinning is regarded as an easily accessible, highly effective and efficient nanofiber fabrication method. One of its significant benefits is the versatility to use a number of types of materials, e.g., organic materials ranging from small molecules to macromolecules, inorganic species and particles, and organic/inorganic hybrids. A typical electrospinning apparatus consists of a high voltage power supply, a spinneret connected to the power supply, and a collector having the opposite charge to the ejection spinneret charge . The fibDespite the outstanding characteristics of electrospinning, improvement to address important issues is in progress by partially modifying the spinning process or even developing new types of spinning process and apparatus. Most notably, the throughput of conventional electrospinning has been a serious bottleneck in its practical applications. Furthermore, it is still challenging to achieve the fabrication of nanofibers with (i) a highly regulated structure, (ii) the desired fiber alignment and orientation, and (iii) three-dimensional complexity in the current electrospinning process.  In the p−1 [In typical electrospinning to fabricate nanofiber mats, a single spinneret is employed because it provides a convenient and cost-effective process. However, it does not offer high throughput which is one of the important requirements for practical and industrial applications. The typical production rate of the single spinneret electrospinning falls in the range of 0.01–0.1 g·h−1 , which h−1 ,28. Howe−1 to 600 g·h−1 which is much higher than that of conventional electrospinning with a single needle [To overcome this issue, needleless electrospinning has been considered as an effective method to maximize the continuity of the fabrication process and therefore, the productivity. Contrary to conventional electrospinning, in the needleless electrospinning technique, an electrical force is applied on the liquid surface directly without using a needle nozzle ,29. To de needle ,46,47. R−1 to 2.25 ± 0.25 g·h−1 . These results highlight the importance of the design of a novel electrospinning spinneret with regard to mass production with high throughput. Niu et al. recently presented a new type of coil-based electrospinning, which uses a secondary coil structure on the coil spinneret, to overcome the aforementioned challenges . The two−1. This achievement is remarkable as the typical productivity of single nozzle electrospinning is in the range of 0.01–0.1 g·h−1 [Lu et al. demonstrated a super-high-throughput electrospinning technique employing an electriferous rotary cone as a spinneret . This te.1 g·h−1 . This elSolution blow spinning which uses concentric nozzles and compressed gas also has attracted attention in nanofiber fields ,53,54,55−1), as well as its thermal, mechanical, and chemical stability [2, to form polymeric foams where multiple polymer jets are ejected from the surface of the foam toward the collector. In the setup, a copper electrode is connected to a funnel to supply electrical power to the polymer solution . In addition, the purity of Nafion nanofiber was about 98%, which has not been achieved with traditional electrospinning. The method was capable of resolving both productivity and purity issues in Nafion nanofiber fabrication.An interesting approach, by Hwang et al., to exploit the foam of polymer solution has recently exhibited improved productivity in Nafion nanofiber fabrication . Nafion tability . Nafion tability ,61. In mtability . Howeversolution a. The apDespite the simplicity and versatility of electrospinning, it has intrinsic limitations for making nanofibers owing to the electrostatic repulsion force that counteracts the surface tension of the droplet. It has recently been demonstrated that the electrical force can be replaced with a mechanical force to fabricate nanofibers. The use of mechanical force overcomes the conventionally accepted limitations of electrospinning, e.g., requirement for high electrical potential and electrically conductive targets to drive the stretching-out of the droplets in the fiber elongation process. In contrast with electrospinning, the use of mechanical force does not limit the type of polymers and solvents because their electrical properties are no longer relevant. Furthermore, it avoids the excessive use of energy in production contributing to high cost. In this section, the efforts on using another type of force than electrical force to make high-quality nanofiber are discussed.As an alternative to electrical force, Lee et al. developed the-so-called handspinning method . It onlyAlthough handspinning offers many advantages, unevenly supplied polymer solution on pulling plates leads to low size uniformity in the resulting nanofibers. This issue was addressed by changing the plate to needle as a tool for drawing the polymer solution, the-so-called needle spinning . Needle The drawing-based technique has been further improved towards a continuous fabrication process using an automated single-step drawing system . The proMost researches conducted with typical electrospinning have demonstrated randomly oriented nanofibers exhibiting a circular cross-section and smooth surface. To expand the applicability of nanofibers, however, constructing particular architectures in two and even three dimensions with structurally well-defined nanofibers is critical. For example, nanofiber patterns with controlled alignment on a two- or three-dimensional substrate were demonstrated for certain purposes ,67,68,69As aforementioned, electrospun nanofiber is generally collected as a nonwoven type due to the uncontrollable nature of jet formation, which limits its applicability. Enormous interest in controlling the nanofiber deposition process has arisen to improve the performance in specific applications. A number of studies dealt with patterning techniques with electrospun nanofiber, including precise positioning, stacking, and orientation ,76, seleRecently, Grilli et al. invented bipolar pyroelectrospinning, which is capable of generating ordered arrays of nanofibers by exploiting periodically poled lithium niobate (PPLN) as a substrate . First, The electrospun jet is highly uncontrollable due to the chaotic whipping jet formation, which makes a randomly orientated nonwoven type nanofiber mat. To suppress the chaotic whipping jet formation and to deposit nanofibers on a substrate with a desired pattern, the near-field electrospinning (NFES) technique has been developed in recent years. NFES provides a straightforward and versatile means to precisely control the location of nanofiber and therefore, to fabricate nanofiber patterns. It features a short tip-to-collector distance less than 3 mm, which leads to a reduction of bending instability of the electrospun jet, and a lower voltage of several hundreds of volts is required. The first proof-of-concept was demonstrated by Lin et al. in 2006 . The NFERecently, Cho et al. successfully fabricated nanofiber stacks built with high-resolution control using the NFES technique  Figure a. In them-phenylene isophthalamide) (PMIA) were collected on a spoked-drum collector for fabricating the aligned and free-standing nanofibers.  in native tissues. However, despite the development of various useful fabrication techniques, building a three-dimensional structure with nanofibers still remains a tough challenge. Widely utilized methods for transforming a 2D structure into a 3D object are the folding, rolling, bending, cutting, and buckling of 2D objects ,93,94. R2 (4%). It also exhibited dramatically rapid sensing speed and strong mechanical bending strength, ensuring the potential as a usable sensor platform. This approach is not complicated for fabricating a flexible and free-standing single-stand yarn scaffold with high-density polymer nanofibers, but it provides different functional core-shell (thread-nanofiber yarn) type fabrics with high flexibility, high surface area, and open porosity.Although electrospun nanofibers have various unique and valuable properties, they are typically known not to be sufficiently mechanically robust to replace common textiles. A few reports have shown that individual electrospun nanofibers can exhibit a reasonable mechanical strength despite their size in nanoscale ,99,100, −1, and the principle for fiber formation does not allow the realization of complex nanofiber structures or patterns. However, other approaches to resolve these issues have recently been proposed. The most important issue is the increase of productivity. The use of multiple nozzles can be the easiest way to do so; however, it brings several issues for processing, and requires an increase of apparatus size, which also increases the cost of equipment as well as of the space. As an alternative, the use of an open spinneret system such as needleless spinning has been shown. This system allows direct application of an electrical force to the liquid surface, generating multiple jets and eventually, achieving an impact on productivity with up to 600 g·h−1. However, it is typically conducted in an open system with a complicated setup requiring the control of many different processing parameters. Consequently, the consistency of the nanofiber morphologies is largely affected, and the complexity in the system negates its applicability in industry. Another issue to have arisen in the spinning technique is the regulation of structure regulation with the control of alignment and orientation. Uniquely designed, but complicated processes and specially outfitted apparatus for patterning or constructing specific structure of nanofibers have been proposed; however, they still suffer from low throughput. Finding an innovative and creative fabrication technique is still ongoing in order to solve all the issues simultaneously to realize high-throughput and easy operation and eventually, to bring the use of nanofibers to the real world. We anticipate that the currently devoted efforts by a number of researchers will eventually bring advanced technologies into this field, with regard to practical implementation in industrial applications. Nanofibers have attracted tremendous technological interest, and related researches are rapidly moving forward to exploit significant characteristics of nanofibers such as high porosity with excellent pore interconnectivity between pores in mats, flexibility with reasonable strength, high surface-to-mass ratio, and the ability to incorporate other materials into actual applications. However, their use in industries have been still challenging due to the poor throughput and productivity of the current fabrication processes, and the difficulties in regulating the structure, alignment, and orientation. In the productivity perspective, electrospinning, one of the popular fabrication techniques, exhibits productivity in the range of 0.01–0.1 g·h


In acute myeloid leukemia (AML), minimal/measurable residual disease (MRD) can be assessed based on molecular markers or immunophenotypic features evaluated at diagnosis, through multiparameter flow cytometry (MFC) for the latter. New artificial intelligence tools allow to perform unsupervised analysis of MFC data. The Flow-Self-Organizing-Maps (FlowSOM) tool was used here to concomitantly compare MFC features of normal bone marrow together with diagnosis and follow-up bone marrow samples from 40 AML patients for the evaluation of MRD. MFC results were compared to molecular MRD, showing high concordance. This opens the road for a new easy and objective way of assessing MRD even in AML patients without molecular markers.The assessment of minimal residual disease (MRD) is increasingly considered to monitor response to therapy in hematological malignancies. In acute myeloblastic leukemia (AML), molecular MRD (mMRD) is possible for about half the patients while multiparameter flow cytometry (MFC) is more broadly available. However, MFC analysis strategies are highly operator-dependent. Recently, new tools have been designed for unsupervised MFC analysis, segregating cell-clusters with the same immunophenotypic characteristics. Here, the Flow-Self-Organizing-Maps (FlowSOM) tool was applied to assess MFC-MRD in 96 bone marrow (BM) follow-up (FU) time-points from 40 AML patients with available mMRD. A reference FlowSOM display was built from 19 healthy/normal BM samples (NBM), then simultaneously compared to the patient’s diagnosis and FU samples at each time-point. MRD clusters were characterized individually in terms of cell numbers and immunophenotype. This strategy disclosed subclones with varying immunophenotype within single diagnosis and FU samples including populations absent from NBM. Detectable MRD was as low as 0.09% in MFC and 0.051% for mMRD. The concordance between mMRD and MFC-MRD was 80.2%. MFC yielded 85% specificity and 69% sensitivity compared to mMRD. Unsupervised MFC is shown here to allow for an easy and robust assessment of MRD, applicable also to AML patients without molecular markers. The prognosis of acute myeloblastic leukemia (AML) has considerably improved in the past few years through progress in therapeutic schedules, emergence of new therapies and a better definition of risk-groups. AMLs are very heterogeneous hematological malignancies, but three prognostic risk groups have been defined by the European LeukemiaNet (ELN) in 2017, respectively with favorable, intermediate and poor outcome, taking into account cytogenetic and molecular criteria . The curMRD detection has become one of the major challenges to adapt therapy and prognosis. Several methods have been developed, allowing to reach very low detection thresholds: reverse transcriptase-quantitative PCR (RT-qPCR), multiparameter flow cytometry (MFC), next-generation sequencing (NGS) and, more recently, digital droplet PCR (ddPCR) . These vMFC has been largely demonstrated to be a mandatory diagnostic tool as well as being quite performing for the follow-up (FU) of any type of AML, independently of molecular anomalies. There is a consensus about the fact that “different-from-normal (DfN)” and “Leukemia-associated immunophenotype (LAIP)” approaches are congruent . The “DfThanks to the huge progress in MFC in the past 15 years, increasing numbers of parameters can now be analyzed simultaneously on small samples. However, information increases in parallel and makes classical bi-parametric interpretation more and more complex. Gating of the blastic population of interest remains highly subjective and operator-dependent as well as the potential observation of subclones. Thus, the major drawbacks of universal MFC analysis of MRD are the increasing number of parameters to take into account and lack of harmonization between panels. In 2018, the ELN produced recommendations, and particularly a combined approach integrating “DfN” and “LAIP” . Simulta®. In this work, we aimed at appreciating the performance of the FlowSOM solution together with Kaluza® in real-life for MRD assessment. This was achieved by comparison to molecular analyses in the evaluation of MRD in 40 AML patients who received a classical intensive chemotherapy treatment. A total number of 96 follow-up (FU) points was analyzed, including FU1 (MRD1 at the end of induction) for all 40 patients.We have previously explored and published ,15 how FFrom the laboratory database of Bordeaux University Hospital , diagnosis data were selected from untreated AML (excluding promyelocytic AML) patients who had been tested between April 2015 and March 2020 and carried a molecular marker allowing for molecular FU. Diagnosis had been performed by morphologic examination according to the WHO classification . Among tIn parallel, as reported previously , 19 NBM NPM1 variant , 6 with RUNX1-RUNX1T1, 4 with CBFB-MYH11 and 1 with BCR-ABL1 . The quantitative detection limit of the assays was 13 copies for NPM1 mutations and 3 copies for fusion transcripts. The achievement of MRD levels below these thresholds was defined as a negative MRD.RNAs were purified from BM mononuclear cells using Trizol reagent . Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit . Molecular residual disease was assessed for escribed ,26. The For each antibody combination, all listmode (lmd) files were processed using the FlowSOM module (Bioconductor version 3.3.2 with flowsom and flocore packages) integrated to the analysis software Kaluza (Beckman Coulter) as reported . In a fiBased on the criteria retained for NOI definition , MRD for® protocol automatically displaying the graphs and statistics described above, compared their results and reached consensus for all time points. All MFC analyses were performed without knowledge of molecular MRD results.Four investigators independently examined all FlowSOM processed samples with a specific KaluzaIn order to compare molecular and MFC results, we chose to consider molecular data as gold standard, which allowed, after combination with MFC values, the definition of True Positivity (TP) when both tests were positive, False Positivity (FP) when MFC was positive in the absence of molecular MRD, False Negativity (FN) in the reverse situation of absence of MFC MRD but presence of molecular MRD and True Negativity (TN) when both tests were negative. This was achieved by building a complex matrix analysis  taking into account MFC NBM and FU levels at all time points compared to the positive or negative status of molecular MRD at the same FU points. The resulting tables were computed with Youden and kappa tests in order to define the best threshold for discriminative sensitivity and specificity of the MCF MRD assessment compared to molecular MRD. This matrix also provided sensitivity, specificity, positive predictive (PPV) and negative predictive (NPV) values.For correlations between patient outcome and FU data, chi square tests were used .Data were available for all samples after MFC analysis with both Tube 1 and Tube 2 panels. Therefore, 192 samples and about 1000 potential MRD nodes were examined.In fine, the median number of positive nodes were, for tubes 1 and 2, respectively 2 (1–6) and 2 (1–7).The global strategy adopted to investigate for MFC MRD was, as described above, to set a tracking gate allowing for node-by-node exploration. Nodes of interest were provided by the NOI strategy, completed in some instances by the exploration of a few nearby nodes in the FREE display. Each retained node had to belong to the area of the Dg blast population, confirmed by backgating on the CD45/SSC histogram displayed together with the Dg MST . Cell nuAs mentioned in the methods section, the optimal concordance between molecular and MFC MRD was determined using Youden and kappa tests instead of ROC curves. The matrix program was run again after adding the molecular data for each FU time point in terms of positivity or negativity. This demonstrated that the thresholds arbitrarily chosen to establish MCF positivity or negativity could be refined to (i) 1.1% (instead of 1%) for the maximal percentage of NBM cells in a given node and (ii) a FU/NBM ratio of 1.9 instead of 2. This optimized threshold only modified previous results for 3 samples.As shown in NPM1-mutated AML and FAB AML4/5. In spite of the classically reported greater difficulty of identifying MFC MRD in these subsets with monocytic differentiation, similar specificities and sensitivities were obtained as compared to the 96 FU time points or 40 FU1 after unsupervised processing.Backgating on CD45/SSC histograms confirmed the similarity between FU and Dg nodes, often disclosing a residual subclone after chemotherapy a,c. Two At variance with what is difficult to identify with classical gating strategies, the FlowSOM unsupervised approach could self-segregate CD56+ and CD56− subclones in the same sample as shown in Finally, in a number of instances, a similar L-shaped pattern was observed for some nodes in the concomitantly tested NBM and FU samples, absent in the concurrent Dg sample . Examinan = 3 cases) with negative molecular MRD  relapsed and died after allogeneic stem cell transplantation (Allo-SCT). For this patient, molecular MRD remained positive until post-AlloSCT. MFC MRD became positive one month before relapse and returned to negativity post-AlloSCT. Conversely, the large majority (N = 29) of CR patients retained both positive post-induction MFC and molecular MRD. Induction failure (more than 5% of blasts in BM at the end of aplasia), noted for 4 patients, correlated with positive MFC MRD for all and with positive molecular MRD for 3. The patient with negative molecular MRD (#9) likely had lost the molecular target (NPM1 mutation) and this discrepancy constitutes a questionable FP MFC MRD.Examination of the outcome of the 40 patients confirmed CR with negative MFC MRD after induction in only 7 cases vs even less  at FU1. Indeed, by testing transcripts, molecular methods may identify fewer cells with large amounts of RNA. Only follow-up of the patients and larger series will ultimately allow to conclude about the best predictor.NPM1 mutations are notably associated with myelomonocytic differentiation [® allows to apply nodes assignment to a mixed population of NBM, Dg and FU lmds which are thus treated in exactly the same way, then to concomitantly display the result of the three MST side by side. This is clearly completely different from the reference to a few  NBM samples reported in many MRD studies. One of the first examples of this indirect comparison was provided by the notion of “empty boxes” [This study demonstrates that an unsupervised analysis of MFC data allows to explore MRD in AML patients with a high confidence in the populations observed. The setting of this work was challenging, since it was decided to include only patients with a possible concomitant molecular FU, known to represent only a proportion of AML patients. Moreover, ntiation . This isntiation . Here, tntiation ,30. The y boxes” ,32. In ty boxes” . Of notey boxes” ,34. HoweAnother advantage of the FlowSOM strategy is of course that the software identifies populations that would have been difficult to select through sequential gating yet can be perfectly characterized during the supervised part of the analysis. To our great satisfaction, FlowSOM allowed to straightforwardly demonstrate concepts taken into account as important in the ELN proposal . As showFlowSOM concomitant analysis of NBM, Dg and FU further allowed to approach the critical issue of significant threshold. As established in the definition of the FlowSOM scripts , we chos−3 threshold [−5 upon acquisition of a sufficient number of cells for negative samples.In our series, the lowest number of FU cells complying with this criterion was 17 cells. This confirms the global opinion of considering clusters of 10–20 cells for limit of detection (LOD) . Of courhreshold  can be iAnother limitation of our study is that we chose molecular MRD as gold standard, raising the question of the significance of FP data, i.e., positive MFC MRD and negative molecular MRD. Nevertheless, the concordance obtained in the different conditions tested  appears quite satisfactory. These types of comparison have been reported in a few cases in the literature with classical MFC analyses. Kern et al. , in a co® software, to evaluate AML MRD. In the challenging set-up of this study, that only considered patients with molecular MRD targets, MFC MRD assessment stood out as a useful monitoring tool. It is thus likely that FlowSOM-assisted MFC MRD assessment, using unsupervised treatment of .fcs files, will be applicable to all AML patients and provide clinicians with useful hints for treatment options.Overall, this study demonstrates the power of the combined unsupervised FlowSOM and Kaluza