Periodontal pockets are spaces or holes surrounded by teeth under the gum line. These pockets can become filled with infection-causing bacteria resulting in tissue, bone, and tooth loss. Cavitation produced by the oscillating tip of dental ultrasonic scalers plays a significant role in routine periodontal therapy to clean these areas. Numerical studies were conducted for a scaler vibrating in a periodontal pocket which was simplified to a hole, using ABAQUS based on the finite element method. The simulations consider the three-dimensional, nonlinear, and transient interaction between the vibration and deformation of the scaler tip, the water flow around the scaler and the cavitation formation. The numerical model was validated by comparing results with experimental data for a scaler vibrating in an unbounded liquid, the displacement at the free end of the scaler and the cavitation pattern near the scaler tip displaying excellent agreement. A parametric study for a scaler vibrating in a hole has been carried out in terms of the volume of the hole, the taper ratio (the radius ratio between the circular opening and bottom of the hole), and the immersion depth of the scaler tip in the hole. The amount of cavitation generated is evaluated by the cavitation density (or the void fraction) which is the ratio of the volume of the cavitation occupied in the hole to the total volume of the hole. Numerical results indicate that the cavitation density in the hole increases with the decreasing hole volume and the increasing taper ratio. It is inferred that cleaning effects could be increased if some modifications to the scaler design could be made to increase the blocking effect of the hole during the cleaning process. Cavitation is observed in the hole even if the scaler is placed above the hole and increases with the immersion depth. These pockets are the perfect area for more plaque to accumulate leading to tooth loss. Dental ultrasonic scalers are used in routine periodontal therapy to clean dental plaque biofilms from teeth and dental implantsCavitation, the formation and collapse of microbubbles in a liquid, occurs in the cooling water around the tips of ultrasonic scalers We will conduct numerical studies for an ultrasonic dental scaler tip vibrating in the water in a periodontal pocket which is simplified to a hole and consider the cavitation generation around the scaler tip. The amount of cavitation generated is evaluated by the cavitation density (or the void fraction), the ratio of the volume of the cavitation occupied in the hole to the total volume of the hole. The numerical simulations can provide an insight into how cavitation may be affected by the hole volume, taper ratio, and immersion depth of the scaler tip. The information will indicate the cleaning effects during the dental cleaning process. The numerical capability can also be used to optimize the scaler design and operation parameters. This study is for the fluid being pure water. Dissolved gases/substances in the solvent will enhance the growth of bubbles due to rectified diffusion. Vyas et al.In the numerical study, the deformation of the scaler tip is modelled by isotropic linear elastic theory, the water flow induced is modelled by the linear potential flow theory in terms of pressure, and their interaction is modelled through the kinetic and dynamic boundary conditions at their interface. Hydrodynamic cavitation is a phenomenon in which the static pressure of a liquid reduces to below the liquid's vapour pressure, leading to the formation of small vapor-filled bubbles in the liquidThe numerical model is described in section 2, and it is validated in section 3 by comparing numerical results with experimental data for the displacement at the free end of the scaler and the cavitation pattern near the scaler tip for a scaler operating in unbounded water. In section 4, the formation of cavitation due to a scaler vibrating in a hole is analysed, and parametric analyses have been carried out to study the effects of the hole volume, the taper ratio, and the immersion depth of the scaler tip on the cavitation generated around the scaler tip in the hole. Finally, the summary and conclusions are outlined in section 5.22.1Oxy-plane with the y-axis vertical. The cubic part of the computational domain is 25 mm×20 mm×20 mm in x,y, and z directions, respectively. The section near the circular cylinder base of the scaler is much thicker than the free end of the scaler and does not deform significantly Asin(ωt) where A is the amplitude and ω is the angular frequency. The choice of the truncated computational domain size is validated in section 3.2. The vibration of the scaler tip generates pressure waves in the water which results in cavitation formation.Considering a dental scaler tip vibrating inside a periodontal pocket (hole) filled with water, as illustrated in Ss = Sfs ∪ tsS where Sfs is the fluid–structure interface within the truncated computational domain, and As shown in 2.2σs and ρs are the stress tensor and density of the structure, respectively, and üs is the second order time derivative of us denoting the acceleration.Suppose the structure  under the action of the body force Introducing an arbitrary variation displacement field Using the Gauss theorem with the gradient identity for the first term on the left side, and neglecting all body forces 2.3Assume that the water flow induced by the vibrating scaler tip is inviscid, compressible and irrotational. The momentum equation of the flow experiencing velocity-dependent momentum losses can be expressed aswith the linear constitutive behaviour of the liquid,To obtain the partial differential equation used in direct integration transient analysis in terms of pressureIntroducing an arbitrary variation field Green’s theorem allows this to be rewritten as.where the boundary traction Two boundary conditions for the fluid part , with the results displayed in 3.2To test if the truncated computational domain is sufficient, we will compare the computational results with two truncated domains, denoted as The results compared are the displacement of the scaler tip and the time-averaged cavitation density in the hole over the simulation time ry close b and the3.3xyz translation stage  and a high-precision rotation mount . The axial rotation of the scaler tip was maintained during experiments.We validate the computational results with experiments for the scaler tip vibrating in an unbounded expanse of water, as there are available experimental results for this case. Experimental high-speed images were acquired of an ultrasonic scaler tip vibrating in a water tank using a previously described setuprwS of the fluid domain in x-direction. For the first few cycles of oscillation, the computational results are associated with some transient oscillations as the initial conditions were set as the quiescent water flow, whereas the experimental data were recorded after many cycles with repeated steady oscillation. After the first few cycles, the displacement of the computational results rapidly stabilizes and agrees well with the experimental data.We compare the computational results with experiments in terms of the displacement of free end of the scaler tip denoted by “+” in 4Parametric study was carried out to investigate the cavitation density in the hole in terms of the hole volume, the taper ratio of the hole, and the immersion depth of the scaler tip. The depth of the holes in our simulations is 10 mm considering advanced periodontitis where the periodontal pocket depth can reach 10 mm or deeper4.1For the scaler tip placed in a hole with rigid boundaries, more ultrasonic energy will be confined in the hole in the form of pressure waves with higher amplitude, and thus the pressure in the liquid is more likely to be lower than the vapour pressure resulting in the higher likelihood of the cavitation formation in the hole.4.2H being 10.0 mm and the radius r being 0.8, 1.5 and 2.0 mm, respectively. The volume of each hole is approximately 20.1, 70.7, 125.7 4.3h/H of 0.50 in holes with the depth H being 10.0 mm and the volume being 70.7 The taper ratio is defined as 4.4H being 10.0 mm, the volume being 70.7 h/H of 0.00, 0.50 and 0.75, respectively.5More cavitation around a scaler tip is generated when it is vibrating in a hole, and the cavitation density increases as the hole volume decreases. This is because the ultrasound energy per unit volume increases inversely with the hole volume.The cavitation density increases with the taper ratio as more energy remains in the hole due to the increased blocking effect. The effects of cleaning should be improved with the addition of a lid added to the scaler tip to cover the hole opening.Cavitation forms in the hole even if the scaler is placed above the hole. The cavitation density increases significantly with the immersion depth of the scale tip in the hole.The cavitation density varies when the scaler tip is put in periodontal pockets with various shapes and immersion depths. The increase of cavitation density may result in increased cleaning efficiency. In this regard, the numerical model developed in this study can aid in further optimization studies that allow the ultrasonic scaler to be used in a non-touching technique in a more efficient way.A numerical study has been undertaken for the cavitation generated by a dental ultrasonic scaler tip oscillating in a periodontal pocket using a coupled acoustic-structural finite element method. As the one-fluid, cut-off cavitation model is employed, cavitation is evaluated by the volume of the region where the pressure falls below the cavitation threshold. The comparison shows that the computational results agree well with experiments in terms of the size and location of the cavitation zones for a scaler tip oscillating in an unbounded expanse of water. The periodontal pocket is simplified to a hole and a parametric study has been performed to investigate the effects of the volume of the hole, the taper ratio You Yu: Conceptualization, Methodology, Formal analysis, Data curation, Investigation, Visualization, Writing – original draft. Mehdi Mahmud: Validation. Nina Vyas: Resources. Warren R. Smith: Supervision, Writing – review & editing. Qianxi Wang: Supervision, Writing – review & editing, Funding acquisition. A. Damien Walmsley: Supervision, Writing – review & editing, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Mentoring is the activity of a senior person (the mentor) supporting a less experienced person (the mentee) in learning. It is based on a trustful, protected, and private atmosphere between the mentor and the mentee. The goal is to develop a professional identity and to reflect on the current situation. At universities, mentors are senior academics or skilled employees while mentees are mostly students with various competencies. Outside universities, mentors and mentees are professionals. In technology-enhanced learning, there is a longstanding tradition of supporting relationships between learners and teachers. Intelligent tutoring systems focus on the cognitive aspects of learning in a selected domain. They have been successfully applied, especially in such areas where the domain of knowledge can be well-formalized with the help of experts. Nevertheless, the learning process is also affected by motivations, emotions, and meta-cognitive competencies, which play a crucial role. In recent studies, these have been recognized and monitored through big educational data and a wide spectrum of available sensors. This enables support for the mentoring process, which is usually spontaneous, holistic, and depends on the needs and interests of the mentee. Psychological and emotional support are at the heart of the mentoring relationship, underpinned by empathy and trust.This Research Topic aimed to investigate relevant aspects of mentoring processes and how they can be technologically supported. The four accepted papers offer various perspectives, ranging from a conceptual analysis of ethical questions to research contributions dealing with concrete scalability tools, novel teaching algorithms as well as policy recommendations in pandemic situations.Köbis and Mehner discuss the relevant ethical questions involved with mentoring and in this way raise awareness of the ethical development and use of future data-driven AI-supported mentoring environments in higher education. They have juxtaposed principles of mentoring ethics and AI ethics with the objective of raising awareness in this interdisciplinary field.Neumann et al. investigate how personal mentoring can be made scalable. They describe the development and implementation of two chatbots that aim to support students of the educational sciences in the self-study of seminar topics and literature. Their results from real-world experiences have the potential to improve the availability of digital mentoring support for all students.Melo and Lopes propose the first machine teaching algorithm for multiple inverse reinforcement learners. Their theoretical analysis shows that teaching a sequential task to a heterogeneous class of learners with a single demonstration may not be possible, as the differences between individual agents increase. They contribute two algorithms that address the main difficulties identified.Batucan et al. explain the factors affecting online learning amidst the COVID-19 pandemic. They empirically test the proposed extended unified theory of acceptance and use of technology (e-UTAUT) model in the students' intention and use behavior toward the online learning system. Insights for higher education institutions and policy directions are recommended.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


KTM2A, previously known as MLL) gene rearrangement KMT2A-r, which is associated with neprilysin (CD10)-negative immature B-cell precursor phenotype and a very poor prognosis [KTM2A-r based on transcription factors (iroquois-class homeodomain protein IRX and homeobox protein HOXA), fusion partners, and corresponding stages of B-lymphopoietic and hemato-endothelial development has been suggested for genomic-driven diagnostics and potential therapeutic strategies in infant ALL [DUX4)-rearranged (DUX4-r), tyrosine-protein kinase ABL (ABL)-class fusions, and myocyte-specific enhancer factor 2 D (MEF2D)-rearranged (MED2D-r) [DUX4-r [Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, in which nearly 5% of the cases are diagnosed before the first year of age . Around fant ALL . Interesfant ALL . Nearly MED2D-r) ,7,8. A s [DUX4-r . Last yeilysin CD-negative [DUX4-r .ALL treatment typically involves several phases: induction , prophylaxis , consolidation ) and maintenance . Targeted therapy protocols including the use of tyrosine kinase inhibitors to Philadelphia chromosome-positive ALL patients and of immunotherapeutic agents such as antibody and chimeric antigen receptor (CAR) T-cell therapies are now being examined to be part of the upfront setting ,13.Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematological cancer. Although most patients with newly diagnosed AML achieve complete remission (CR) after intensive induction and consolidation therapy, more than half of them relapse within the next three years ,15. BecaFLT3 mutations), combined epigenetic therapy with the BCL-2 inhibitor venetoclax, immunotherapy or restricted approaches for AML with myeloid-related changes (AML-MRC), or therapy-related AML (CPX-351) cases [Besides intensive induction and consolidation therapy, AML treatment has improved in recent years, and several new therapeutic options have been approved. Most of them include mutation-specific approaches  cases .The genetic and clinical heterogeneity of ALL and AML cases represent a challenge even for the new therapies. Moreover, relapse and treatment resistance seriously hinder ALL and AML treatment. To understand the biology underlying those differences, omic sciences have increasingly been utilized to produce useful knowledge.https://proteomics.se/forall accessed on 8 January 2023) identified the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the myocyte enhancer factor 2D (MEF2D)-heterogeneous nuclear ribonucleoprotein U-like protein 1 (HNRNPUL1) fusion high-risk subtype, for which this drug activates pro-apoptotic mitogen-activated protein kinase (ERK) signaling associated with the molecular mediators of pre-B-cell negative selection [Multi-omic analyses of 49 childhood ALL cell lines using proteomics, transcriptomics, and pharmacoproteomic characterization , at IDH1/2 mutations displayed a high expression of the 2-oxoglutarate–dependent histone demethylases KDM4A/B/C, despite no changes in messenger RNA levels for these genes, and samples with FLT3-tyrosine kinase domain (TKD) mutationsassociated with the activation of the SRC-family tyrosine kinases FGR and HCK) [To uncover the molecular changes allowing AML cells to escape treatment, two proteomic studies with liquid chromatography–mass spectrometry (LC–MS) and serial time-point samples during the disease progression of patients have shown that the proteomic profile at relapse is enriched for mitochondrial ribosomal proteins and subunits of the respiratory chain complex, indicative of reprogrammed energy metabolism from diagnosis to relapse ,24. Usinand HCK) . Using sand HCK) .Altogether, these studies show us the importance and strength of single- and multi-omic approaches to decode the complex and heterogenous molecular basis of acute leukemias and to improve the current treatment and survival of ALL and AML patients.https://github.com/j-rehn/RaScALL accessed on 10 January 2023) [In the past few decades, single-omic strategies have been used for research leading to ALL- and AML-related biomarker discovery. Genomic approaches have described several causative variants that are now part of international diagnosis guidelines. However, the integration of genome sequencing into a healthcare setting still lags behind. Nevertheless, examples of genomic incorporation, such as the reported initiative across multiple clinical entities of rare diseases  and the ry 2023) .Numerous single-omic studies with patient cohorts have revealed a high number of leukemia biomarkers ,30,31. RMulti-omic approaches that integrate the data from distinct levels of the cellular organization, i.e., from genes to metabolites, have recently been reported, which offer new perspectives for innovation in ALL and AML diagnostics and therapeutics. Multi-omic strategies bring together researchers with different expertise, including data managers and bioinformaticians, to provide protein and metabolite targets of specific genomic backgrounds. The incorporation of more clinicians in research teams appears to be more necessary than ever to transfer clinical innovation into practice.


Hanseniaspora uvarum, Meyerozyma guilliermondii and Pichia kudriavzevii using 5:1 ratios (Kveik vs. NCY) at 20 °C. The combinations of Kveik No 1 with P. kudriavzevii and Kveik No 1 with Hanseniaspora uvarum showed substantially increased amounts of specific volatile aroma compounds that were previously identified in the NCYs. Our results indicate that Kveik isolates appear to be suitable for co-fermentations with certain NCY to enhance beer or spirit fermentations, increasing the potential of these yeasts for beverage productions.Kveik are consortia of yeast used for farmhouse ale production in Western Norway. Yeast strains derived from these mixtures are known, for example, for their high fermentation rate, thermotolerance, lack of phenolic off flavor production (POF-) and strong flocculation phenotype. In this study, we used five single cell yeast isolates from different Kveik yeasts, analyzed their fermentation and flavor production, and compared it with a typical yeast used in distilleries using 20 °C and 28 °C as the fermentation temperatures. One of the isolates, Kveik No 3, showed an impairment of maltotriose utilization and thus a reduced ethanol yield. Kveik fermentations for spirit production often harbor bacteria for flavor enrichment. We sought to improve Kveik fermentations with non-conventional yeasts (NCY). To this end we co-fermented Kveik isolates with  Saccharomyces have a long history of domestication due to their constant use in baking and alcoholic beverage production [S. cerevisiae may be considered mankind’s first domesticated species [S. cerevisiae and S. eubayanus, and today two main groups are recognized as Saaz and Frohberg, which evolved from a single hybridization event [Yeasts of the genus oduction . Accordi species . Domesti species . Lager yon event ,4. Theseon event ,6. Lageron event .S. cerevisiae yeasts that cannot utilize melibiose in contrast to lager yeasts and ferment at higher temperatures (15 °C–25 °C) due to higher maltose transport activity at these temperatures [Ale yeasts, on the other hand, are mainly eratures .A group of farmhouse ale yeasts, used for centuries in an isolated region of Western Norway, are known as Kveik . Kveik rS. cerevisiae yeasts that belong to the ‘Beer 1’ clade composed of yeasts from Germany, the UK and the US. However, they also contain a S. cerevisiae hybridization partner that is yet unknown [MAL genes, POF- phenotype based on loss-of-function mutations in either PAD1 or FDC1 required for the decarboxylation of hydroxycinnamic acids to 4-vinylguaiacol, and a high flocculation rate based on copy number variations in the flocculation genes FLO1 and FLO11 [S. cerevisiae and S. uvarum, indicating that some degree of diversity can be expected [A comparative genome analysis of six Kveiks indicated that they are tetraploid  unknown . Severalnd FLO11 . The accnd FLO11 . One isoAs Kveiks adapt to high temperature fermentations, they have a strong potential to be used in a variety of fermentations that then are sufficient without cooling. Besides energy cost saving, faster fermentation speed of Kveiks may also free up capacity in breweries. Kveiks  have been used in stout beer and Indian Pale Ale fermentations and have generated more fruity aromas than a commercial control strain ,13.Saccharomyces in fermentations, compared to some successful spontaneous fermentations that are based on a much broader biodiversity, have led to an interest in employing NCYs in beverage productions. Studies on NCYs have been increasing over the past two decades with a focus on lowering the ethanol content and/or enrichment of flavors in the final beverage [Wickerhamomyces anomalus in co-fermentations with S. cerevisiae strains to enhance the flavors in beer and wine [Non-sterile fermentations, such as in whisky production, require fast fermenting strains with high temperature and high ethanol tolerance. The presence of bacterial strains is tolerated and may be associated with positive aroma contribution ,15. The beverage ,19,20,21and wine ,23.H. uvarum, M. guilliermondii and P. kudriavzevii as potentially beneficial strains for co-fermentations [Previously, we isolated and characterized a series of NCYs and identified ntations . In thisYeast strains were subcultured in YPD . The YPD plates for single colony isolation using the streak plate method contained 2% agar. Unhopped and rehydrated granmalt  was prepared at 15° Plato and sterilized by autoclaving. The same batch was used for all experiments. The sugar content of the malt was determined by HPLC (High-Performance Liquid Chromatography) and is shown in For sporulation, assays cells were precultured in liquid YPD, centrifuged and washed with sterile water and then streaked out on solid sporulation media . Plates were incubated for 10 days at 25 °C before determining the sporulation efficiency. The strains used in this study are outlined in Microscopic images of Kveiks and ale yeasts were generated using Differential Interference Contrast (DIC) on a Zeiss AxioImager M2  equipped with a Plan-Apo 100x/1.4 Oil objective. Images were acquired with a pce.edge 4.2 bi digital camera and processed using Fiji software  .Micromanipulation of yeast tetrads was carried out on an MSM System 400 . A loopful of cells from a sporulation plate were resuspended in 45 µL sterile water and treated with 5 µL zymolyase (10 mg/mL) for 30 min at 30 °C. A portion of 10 µL was streaked out on the inoculation field for tetrad dissection.7 cells/mL. Fermentation temperatures were kept constant at either 20 °C or 28 °C. Fermentations were stirred constantly at 300 rpm. The fermentation progress was monitored by documenting mass loss due to CO2 release. Co-fermentations of Kveiks with NCYs were carried out at 20 °C. The fermentations were carried out with initial ratios of one part NCY and five parts Kveik. Appropriate dilutions were generated based on OD600 nm measurements. All fermentations were carried out in triplicate.Lab scale fermentations were carried out in 250 mL tall-tube cylinders filled with 150 mL Granmalt wort. Wort was pitched with yeasts at 1 × 102 release reached a plateau. Sugar consumption and residual sugar concentrations were measured by HPLC at the end of fermentation as described [The end of fermentation was reached when mass loss and COescribed ,26. Volaescribed .To obtain estimates of relative effect sizes of (A) the Kveik strain and (B) the NCY strains on volatile aroma composition, we performed redundancy analysis (RDA) on the VAC data set see . StatistKveiks are described as farmhouse ale yeasts. To compare their life cycles with ale yeast production strains we used five Kveiks see ; we geneThe ale yeasts that were studied showed quite a variable degree of sporulation, but sporulation can reach a very high level, as seen in W174 . Nevertheless, the germination efficiency of ale yeast spores was surprisingly low, making ale yeasts generally difficult breeding stocks. Kveiks, on the other hand, sporulated quite well and the germination efficiency was considerably higher, as has also been observed by others . Kveik 5Kveiks are able to ferment rapidly at high temperature. However, in other brewing settings lower temperatures are employed. Thus, we studied the fermentation progress and the sugar utilization of Kveiks at 20 °C and 28 °C. Kveiks showed strong fermentation intensities at the start of fermentation and finished fermentation a day earlier at 28 °C, compared to the 20 °C fermentations A. FermenA complete utilization of available sugars is required for maximal ethanol yield. We quantified wort sugars  using HPLC. This indicated that Kveiks, particularly Kveik 3, did not fully consume maltotriose. While Kveik 3 consumed only 25–30% of maltotriose at the tested temperatures, the other Kveiks utilized between 81% and 91% of the maltotriose. The M1 strain outperformed the Kveiks in terms of maltotriose utilization. However, M1 could not consume all of the maltose at a higher temperature (only 88%), in contrast to the Kveiks. As maltose is the major wort sugar, overall sugar consumption was higher in the Kveiks, except for Kveik 3. The amount of ethanol produced by Kveiks was in the expected range, yet, was found to be lower than that of M1. Overall, the ethanol yield was higher at 20 °C than at 28 °C .Our fermentations are routinely analyzed for the major esters and higher alcohol volatile aroma compounds. Fermentations of Kveiks at 20 °C were compared with M1 fermentations. The most abundant ester by far in these fermentations was ethyl acetate (acetic acid ethylester) in the range of 37 to 95 mg/L. M1 produced almost twice as much ethyl acetate as the Kveiks. The other major esters were isoamyl acetate (acetic acid 3-methylbutylester), ethyl octanoate (octanoic acid ethylester) and 2-phenyl acetate (acetic acid phenylethylester), which made up 67–84% of all other esters in these strains.Kveik 3 and M1 were similar in their isoamyl acetate production, but overall, the Kveiks generated substantially less esters compared to M1, with Kveik 1, 2 and 4 reaching around 60% and Kveik 5 about 75% of the levels found in M1. Kveik 3 was different in that it produced a high amount of isoamyl acetate and 2-phenyl acetate, which are both products derived from Ehrlich pathway degradation of amino acids leucine and phenylalanine, respectively see .Although M1 outperformed the Kveiks in the production of esters, the profiles for higher alcohols  were very similar in all strains and amounted to a total of 250–320 mg/L, but still with M1 at the higher end. The major acids detected were i-valeric acid and decanoic acid .In fermentations at 28 °C, M1 again produced the highest amount of ethyl acetate, ~twice as much as Kveiks 1, 4, 5 and ~50% more than Kveiks 2 and 3. However, the amount of other aroma esters generated by M1 was considerably lower at 28 °C compared to the 20 °C fermentations (only 40%), suggesting lower production or retention at higher fermentation temperatures. The Kveiks performed quite heterogenously. Kveik 1 was similar to M1 in that it showed an overall reduction of ester compounds at 28 °C, compared to 50% of that at 20 °C fermentations. Kveik 2, on the other hand, performed at 28 °C: very similar to the 20 °C fermentations. The only major change was a reduction in isoamyl acetate production in this strain, which was contrasted by an increased ethyl decanoate (decanoic acid ethylester) production. Kveiks 3, 4 and 5 showed smaller reductions in ester compounds: 76%, 83% and 95% of the 20 °C levels, respectively, also with substantial reductions in isoamyl acetate production .The production of higher alcohols at 28 °C was very similar to the production at 20 °C. Interestingly, under these conditions M1 was found at the lower end of the scale (240 mg/L). The major higher alcohol at both fermentation temperatures was isoamyl alcohol, accounting for ~50% of the higher alcohols. The amounts of isovaleric acid were similar under both fermentation conditions  generated substantially higher amounts of esters (except ethyl acetate) than M1. However, overall production of esters was improved at lower temperatures in all strains and M1 outperformed the Kveiks at 20 °C. Therefore, we aimed for a targeted improvement of Kveik flavor output by adding NCYs in co-fermentations. Previously, we characterized three NCYs, ntations . These yntations . As thesH. uvarum co-fermentation where a residual 3% of the maltose was found at the end of the experiment; see 2 was released compared to Kveiks 1,2 and 4, which was also reflected in their lower ethanol yields .The M1 strain performed well in all co-fermentations. With M1-As Kveik 3 and Kveik 5 did not perform satisfactorily, we focused on a comparison of aroma profiles of co-fermentations of Kveiks 1, 2 and 4 with those of M1. In these co-fermentations we aimed for an increase in propanoic acid ethylester, butyric acid ethylester and i-butyric acid ethylester via the NCYs, which actually could be demonstrated .P. kudriavzevii example up to five-fold. However, the ethyl acetate production remained at a much lower level than seen before in NCYs’ single fermentations [H. uvarum and the P. kudriavzevii generated higher amounts of 2-phenyl ethanol.The M1-B143 co-fermentations revealed that flavor in this distillery strain could also be modulated in this targeted way. The co-fermentations led to an increase in ethyl acetate production, e.g., in the Kveik 1—ntations . Co-fermP. kudriavzevii co-fermentations produced a substantial increase in volatile aroma compounds over the Kveik 1 single fermentation, which can mostly be attributed to the input of the desired NCYs esters. However, the generation of flavor compounds based on amino acid metabolism through the Ehrlich pathway was negatively affected in most co-fermentations [P. kudriavzevii showed the overall best results with increased ethanol and aroma ester production, outcompeting all other strains, including M1, at 20 °C. The runner-up in these trials was the Kveik 1 co-fermentation with H. uvarum , ferment well at high temperatures(>30 °C), tolerate high ethanol levels, produce no phenolic off-flavors, flocculate abundantly and tolerate drying and dry storage . Kveiks Ale yeasts may sporulate abundantly but show a rather low spore viability . The pooKveiks will now certainly be tested in all different brewing and baking environments, and first reports on Kveiks used for Indian Pale Ale and Stout beer and wine fermentation have been published ,13,23. OLactobacilli may contribute beneficial flavors to whisky [Aroma compounds may enter whisky upon distillation. Therefore, yeasts that generate copious volatile aromas may be favorable. Flavor contributions of other organisms have been viewed as spoilage, but recently they have also been looked at more positively ,32. The o whisky ,33. Parto whisky ,34. Howeo whisky . This suS. cerevisiae, e.g., for lowering final ethanol content, for contributing aroma-related compounds or antioxidant activity, for producing craft beers or low-calorie beers, and to enhance glycerol production [Brettanomyces, Hanseniaspora, Kluyveromyces, Pichia, Meyerozyma, Torulaspora or Zygosaccharomyces [H. uvarum was tested in co-fermentations of grape must with S. cerevisiae, while P. kudriavzevii was shown to have some potential to co-ferment an overripe Cabernet Sauvignon grape must [NCYs have been employed in different co-fermentation regimes with oduction ,38,39,40aromyces ,42. H. uape must ,44. As wp = 0.01), respectively  and less for the Kveik strains  use of high-gravity wort to increase acetate ester production ; (ii) thSaccharomyces co-fermentations. In particular, our study opens the way for larger scale trials using the specific strain pairs Kveik 1/H. uvarum and Kveik 1/P. kudriavzevii in co-fermentations.We have characterized Kveiks in wort fermentations and we were able to improve ester compound formation by the co-fermentation of Kveiks with NCYs. This represents a successful example of targeted flavor manipulation in a fermented beverage using specific pre-selected NCYs. The NCYs impacted the flavor output independently of the Kveiks tested. It is thus likely, that these NCYs will be suitable also in a wider range of


Drosera have evolved leaf movements and enzyme secretion to facilitate prey digestion. The molecular underpinnings of this behavior remain largely unknown; however, evidence suggests that prey-induced electrical impulses are correlated with movement and production of the defense hormone jasmonic acid (JA), which may alter gene expression. In noncarnivorous plants, JA is linked to electrical activity via changes in cytoplasmic Ca2+. Here, we find that dynamic Ca2+ changes also occur in sundew (Drosera spatulata) leaves responding to prey-associated mechanical and chemical stimuli. Furthermore, inhibition of these Ca2+ changes reduced expression of JA target genes and leaf movements following chemical feeding. Our results are consistent with the presence of a conserved Ca2+-dependent JA signaling pathway in the sundew feeding response and provide further credence to the defensive origin of plant carnivory.Some of the most spectacular examples of botanical carnivory—in which predator plants catch and digest animals presumably to supplement the nutrient-poor soils in which they grow—occur within the Droseraceae family. For example, sundews of the genus  Drosera just like a living creature, and I suppose he hopes to end in proving it to be an animal”  , to which animal prey adheres. Stimuli from the captured animal generate action potentials along the tentacle and cause oscillations in membrane potential of the local leaf blade , 4. Thesovie S1) .Arabidopsis thaliana (Arabidopsis), mechanical wounding by herbivorous insects generates systemic electrical signals that coincide with waves of increasing cytoplasmic Ca2+ concentration ([Ca2+]cyt) that drive JA production . Nevertheless, many leaves responded to feeding, allowing us to explore [Ca2+]cyt changes in these plants.To image [Car GCaMP3  using biDrosophila melanogaster flies to leaves caused dynamic [Ca2+]cyt changes. Most prominent was increased GCaMP3 fluorescence and flickering in tentacles, observed in 19/20 experiments (Movie S2). The increase in GCaMP3 intensity decreased over time and was gone within 30 min, even if the insect remained trapped and continued struggling (n = 8). Tentacles under or in close contact to the fly sent a Ca2+ wave from the tentacle head down to the base, followed by relatively low-intensity, slow-moving waves that propagated outward from the base . These changes correlate with the top-down propagation of action potentials in the tentacle and oscillations in leaf blade membrane potential following head stimulation recorded by others (2+ waves (Bottom and Movie S4).Feeding of live y others . In addin = 30, both treatments; P < 0.005, Fisher’s exact test). We therefore assessed the effect of mechanical touch on [Ca2+]cyt. When bent, 84% (52/62) of tentacles showed changes in GCaMP3 intensity, with Ca2+ signal increasing throughout the stalk or near the base (Movie S5). Similarly, a gentle manual touch of a glass probe onto the leaf caused transient GCaMP3 increases similar to those caused by live prey . However, hard touch with some lateral probe movement initiated one or more intense Ca2+ waves that propagated rapidly to distal leaves (Movie S7). The average speed of the first systemic wave was 1.6 mm/s, similar to those in Arabidopsis following wounding , suggesting that inflection is due to salt treatment and not mechanical placement of the liquid droplet onto the leaf. Over 3 h, salt treatment  also induced one or more systemic Ca2+ waves in four of five treated leaves (Movie S8). These occurred 2 min to 70 min following treatment, with a speed similar to those induced by hard touch , and were not observed in water-treated controls (n = 5).To assess the effect of chemical stimuli, we fed salts to the leaves. A 5-μL drop of 5× Murashige and Skoog basal salts with vitamins (MS) solution placed on the leaf blade caused leaf inflection of 90° or more in 15 of 30 leaves in a 12-h period. By contrast, 0/30 water-treated controls inflected  and OXOPHYTODIENOATE-REDUCTASE 3 (OPR3)   were ind3 (OPR3) A and B. et genes C. Strikis tested C. Lastlye effect D. Togeth2+ changes in carnivorous sundew plants. These changes are both local and systemic in nature and precede leaf movements. How the plant interprets and integrates these and other Ca2+ signals to generate an appropriate response remains an outstanding question.Here, we report prey capture–associated dynamic cytoplasmic CaArabidopsis also elicit touch-induced Ca2+ signals. For example, trichomes may sense mechanical stimulation from falling rain droplets to defensively prepare against rain-associated pathogen invasion (Interestingly, leaf trichomes of invasion , or deteinvasion . It is t2+ signals is consistent with a model whereby Ca2+ oscillations alter JA production (Finally, our results further advance the notion that carnivory in the Droseraceae evolved from the JA insect defense pathway . Our finoduction E. With t35S promoter:GCaMP3 plasmid. From this, a transgenic reporter line was regenerated and clonally propagated in tissue culture before moving to soil. Clonal Ca2+ reporter plants were imaged with fluorescent dissecting microscopes. All other experiments used wild-type plants. For qRT-PCR, SYBR Green-based methods were used. See SI Appendix for details.Callus generated from sundew leaf explants was bombarded with gold particles carrying a 2×Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Marajó Island, within in the Amazon River Delta, supports numerous bands of feral equids including the genetically distinct Marajoara horses. Approximately 40% of the equids on the island are infected with Equine infectious anemia virus (EIAV). This high seropositivity rate coupled with the need to preserve rare breeds such as the Marajoara horse precludes euthanasia as the primary means for controlling EIAV in this region. In the absence of iatrogenic transmission, spread of this lentivirus is mediated primarily by hematophagous insects, whose year-round prevalence on the island is supported by favorable climatic conditions. In addition, cases of vertical EIAV transmission have been observed suggesting inclusion of seropositive mares in restorative breeding programs could result in their progeny becoming infected with this virus either pre-parturition or post-partum via hematophagous insects. Therefore, the aim of this study was to evaluate EIAV vertical and post-partum insect-mediated transmission rates among foals born to seropositive feral mares until natural weaning. Serum samples from foals born to seropositive feral mares within the Soure municipality, of Marajó Island, were collected to investigate their serological status, using an indirect ELISApgp45, with positive samples confirmed using the classical agar gel immunodiffusion (AGID) assay.The serological status of 28 foals were monitored over a 2-year period with some subjects, depending on their date of birth, being sampled up to six times. All foals remained with their respective mares until fully weaned at approximately 10 months of age. Only 2 foals (7.14%) in the study group became seropositive against EIAV.The results demonstrate that in most cases it is possible to obtain seronegative foals born to and eventually weaned by EIA positive mares, even in equatorial regions where substantial rainfall and high temperatures favor the proliferation of insect vectors. Marajó Island (Pará state), situated in the Amazon River delta, is the world’s largest fluvial land mass and home to approximately 30,000 equids . Among tThe initial horse population was interbred with some other Portuguese breeds, including Arabian and Alter Real, and eventually, became recognized as the distinctive Marajoara horse. One of the major characteristics of this breed is adaptation to adverse tropical conditions , 6. AlthConsequently, the equid population of Marajó Island contains distinctive, rare horse breeds that should be preserved, because they possess genetic characteristics uniquely associated with adaptation to the tropical environment of the Amazon River Delta. However, in addition to dilution of these genetic characteristics by interbreeding, another major threat to equids inhabiting Marajó Island is Equine Infectious Anemia Virus (EIAV). In a survey conducted on the island of just 294 equids, 46.26% were found to be seropositive for this virus  and 2 mules [Equus caballus x Equus asinus]) born to mares that were or became seropositive for EIA. Although the majority of the horse foals were of mixed breed (n = 18 or 64.3%), eight subjects (28.6%) were Marajoara horses. Of the 388 equids on the property 75 were used for livestock management while an additional 54 were either in training for livestock management or being prepared for sale. However, the majority (259), including those equids used in this study were not confined to pastures or subject to any form of conventional animal husbandry. Therefore, they were defined as living a “feral” existence, in being free ranging within their environment and, except for sample collection, not subject to regular human interaction, including the administration of vaccines or dietary supplements. Within the feral equid group parturition always occurred under natural conditions and foals remained with their mothers until completely weaned, which normally occurred at approximately 10 months of age.The study period was from January 2018 until January 2020 and involved 28 foals  guidelines.Antibodies to the EIAV gp45 transmembrane envelope glycoprotein were detected by indirect ELISA (ELISApgp45) as described by Naves et al. (2019) . This asAll ELISApgp45 positive samples were re-tested using a commercially available AGID test kit, according to the manufacturer’s recommendations , to confirm the results. As recommended by the Brazilian regulatory authorities, only serum samples that reacted in both tests were classified as positive .During the course of this study, blood samples for serological analysis were collected from 26 horse and 2 mule foals. However, the foals used in this study were of different estimated ages, with half of the study group being born after first sample collection date in January 2018. Hence, the foals were divided into three groups. Group A consisted of 14 foals born prior to January 2018 Table . At the Two foals in Group B 398 and 422) produced test positive results at only the March 2018 and July 2018 sample collection points , for example, it has been demonstrated the risk of vertical transmission from mother to neonate in the absence of antiretroviral drug therapy ranged from 15 to 35% . MoreoveAlthough studies in horses are far from exhaustive, it appears that EIAV can be transmitted in utero with an incidence of 100% (7/7) if the mares experience a clinical episode during gestation. On the other hand, the probability of vertical transmission decreases to less than 11% (5/45) if the mare remains asymptomatic. It is important to note that the 11% of foals born to asymptomatic mares did not develop clinical signs until 4 months of age and were maintained in close proximity to an EIAV seropositive foal. Therefore,in these cases transmission may not have originated from the respective mares, but could have occurred horizontally via the infected foal through insect bites . RegardlAlthough vertical transmission of EIAV to foals born to inapparent carrier mares appears to be unusual, they may still be exposed to the virus via the feeding behavior of hematophagous insects, especially as the mare and foal are by definition frequently in very close proximity until weaning is complete. To reduce the risk of insect mediated transmission, Silva et al. 2001) removed foals born to EIAV seropositive mares at 6 rather than 10 months of age and segregated them from the rest of the herd by at least 200 m . In a st01 removeCollectively, these results demonstrate that of 28 foals studied, just 2 (7.14%) became infected by EIAV. This is in agreement with a survey conducted in the Pantanal Sul-matogrossense region of Brazil, where seropositivity rates in foals approximately 1 year of age were found to be only 3.3%, despite 65.93% of the general equid population being serologically test positive for EIAV . AlthougAlthough colostral antibodies are usually detectable in serum of foals born to EIAV seropositive mares, to our knowledge, the efficacy of these at preventing EIAV infection has not been investigated. Besides that, another protective factor might be the fact young foals have a more vigorous defensive response to the presence of hematophagous insects than adult equids. These defensive movements often cause the insect to relocate to a more passive host. It has for example been observed that in freely grazing horse herds, foals have only 2.43% the tabanid burden found on older animals . In addi6 horse infectious dose 50 (HID50) per mL of peripheral blood. The mouthparts of a large hematophagous insect, such as Tabanus fuscicostatus, retain 10 ± 5 nL of blood after an interrupted blood meal that in turn should, in the case of feeding on febrile equids, equate to 5 to 15 infectious EIAV particles. This suggests that, in theory, spread of EIAV can occur following a single fly-bite on an equid with clinical signs of EIA although experimentally it has been shown that transmission by T. fuscicostatus under these circumstances occurred in 1 of 7 attempts. In contrast, inapparent carriers have viremia titers of 1 HID50 per ml of peripheral blood or less. Under these circumstances, the numerical probability of transmission by T. fuscicostatus following an interupted blood meal is approximately 1 in 67,000–200,000 [Although antibody transfer in colostrum and the more aggressive defensive behavior to hematophagous insects may contribute towards the low incidence of EIAV infection reported in this and other studies , 36, blo–200,000 –41. If tIn conclusion, evidence is presented that supports the argument that vertical transmission of EIAV is an infrequent event in foals born to asymptomatic EIAV seropositive mares. Moreover, insect mediated transmission rates from inapparent carrier mares to foals are low thereby, enabling a foal to remain with its mother until fully weaned, even in conditions that are highly conducive for maintaining year-round high density insect vector populations. These findings demonstrate that it is possible to obtain EIAV-free foals, even when born to seropositive mothers providing the mares do not have clinical signs of EIA. This information is important for preserving genetically valuable equid traits, such as those found in the rare Marajoara and Puruca horse breeds that are currently threatened with extinction.


BCO2 genotype and the addition of Aztec marigold flower extract to rabbit diets on the expression of BCO1, BCO2, LRAT, and TTPA genes in the liver. The levels of lutein, zeaxanthin, β-carotene, retinol, and α-tocopherol in the liver and blood serum of rabbits, as well as plasma biochemical parameters and serum antioxidant enzyme activities were also determined. Sixty male Termond White growing rabbits were divided into three groups based on their genotype at codon 248 of the BCO2 gene ; each group was divided into two subgroups: one subgroup received a standard diet, and the other subgroup was fed a diet supplemented with 6 g/kg of marigold flower extract. The obtained results indicate that the BCO2 genotype may affect the expression levels of BCO1 and BCO2 genes in rabbits. Moreover, the addition of marigold extract to the diet of BCO2 del/del rabbits may increase the expression level of the BCO2 gene. Finally, an increase in the amount of lutein in the diet of rabbits with the BCO2 del/del genotype contributes to its increased accumulation in the liver and blood of animals without compromising their health status or liver function.This study investigated the effect of the  BCO2 gene gives a yellow tint to fat [Carotenoids constitute a lipophilic group of isoprenoid pigments . They art to fat . The yelt to fat ,29,30.Tagetes erecta L.) belongs to the family Asteraceae and is native to Mexico. It is an annual plant grown in Europe, Asia, and Africa for ornamental, medicinal, and industrial purposes [Marigold flowers are a valuable source of carotenoids. The Aztec marigold  in the liver. The levels of lutein, zeaxanthin, β-carotene, retinol, and α-tocopherol in the liver and blood serum of rabbits were also determined, as well as plasma biochemical parameters and serum antioxidant enzyme activities.Our previous studies analyzed the levels of carotenoids, retinol, and α-tocopherol in the adipose tissue and liver of rabbits and in the milk of rabbit depending on their genotype determined by AAT-deletion mutation at codon 248 of the  animals  and that animals , this stBCO2 gene was associated with changes in the expression of the BCO1 gene in rabbits fed the control diet . An intermediate level of BCO1 gene expression was noted in rabbits with the deletion of both BCO2 alleles. Genotype at codon 248 of the BCO2 gene did not induce changes in the expression of the remaining genes in rabbits fed the control diet.Genotype at codon 248 of the rol diet . The relBCO2 gene was associated with changes in the expression of the BCO2 gene in rabbits fed a diet supplemented with 6 g/kg of Aztec marigold flower extract . An intermediate level of BCO2 gene expression was noted in heterozygous rabbits. Genotype at codon 248 of the BCO2 gene did not induce changes in the expression of the remaining genes in rabbits fed the marigold-supplemented diet.Genotype at codon 248 of the  extract . The relBCO2 gene  than in those with ins/ins and ins/del genotypes (p < 0.01 in both cases). Moreover, the addition of marigold extract to the diet of rabbits with the del/del genotype caused a four-fold increase in their liver lutein levels (p < 0.01), compared with their counterparts fed the control diet. Differences were also found in β-carotene concentrations in the liver of rabbits. In the control group, the levels of this micronutrient were higher in homozygous del/del rabbits than in those with ins/ins and ins/del genotypes (p < 0.05); in the marigold group, a significant difference was noted between rabbits with the del/del genotype and those with the ins/del genotype .Liver lutein concentrations were many times higher in rabbits with double deletion at codon 248 of the enotypes . The aboBCO2 genotype affected the serum levels of lutein and β-carotene (p < 0.01 in both cases). The same relationships were observed for β-carotene levels in the groups fed both diets (p < 0.01 in both cases). Moreover, the diet affected the serum concentrations of lutein, zeaxanthin, and β-carotene. Rabbits with the del/del genotype fed the marigold-supplemented diet were characterized by higher levels of lutein and zeaxanthin than their counterparts fed the control diet (p < 0.05 in both cases). A similar relationship was noted for β-carotene content in homozygous rabbits (p < 0.05).The carotene . Lutein BCO2 gene polymorphism affected the plasma levels of ALP and LDH (p < 0.01). Plasma LDH levels were lower in homozygous del/del rabbits than in animals with the ins/ins genotype (p < 0.01 in both groups). Rabbits with the del/del genotype fed the marigold-supplemented diet had lower LDH levels than their counterparts fed the control diet . and LDH . In the p = 0.070).No significant differences were found in the activity of antioxidant enzymes in the blood serum of rabbits . In the BCO2 gene differentiates lutein content in the liver and carotenoid concentrations in perirenal fat in various crossbred rabbits. However, the question whether the dietary inclusion rates of carotenoids affect their accumulation in the body remained unanswered. To the best of our knowledge, this is the first study involving rabbits with various genotypes at codon 248 of the BCO2 gene fed diets with different inclusion levels of carotenoids. Specifically, the control group received 21.64 mg/kg of lutein, whereas the marigold group received almost 320 mg/kg of lutein. Such an approach allowed us to determine changes in the expression of genes related to the metabolism of carotenoids and vitamins A and E in the liver (1), the rates of carotenoid, retinol, and α-tocopherol accumulation in the liver and blood serum (2), and blood plasma biochemical parameters and the activity of antioxidant enzymes (3).The present study investigated the expression of genes related to the metabolism of carotenoids and vitamin A with regard to the excess of carotenoids in feedstuffs offered to male Termond White rabbits. It was hypothesized that the surplus amounts of lutein, zeaxanthin, β-carotene, retinol, and α-tocopherol will not affect the health status of the host or liver function. Previous research  has showBCO2 gene were characterized by higher expression of the BCO1 gene than the animals with the ins/ins genotype. A similar tendency in BCO1 expression, although not statistically significant, was observed in marigold group rabbits. This phenomenon can be attributed to the role played by the BCO1 gene. Specifically, BCO1 is a β-carotene cleavage enzyme leading to the formation of vitamin A. Thus, it is not surprising that in this study, the increased level of BCO1 expression in animals with the ins/del genotype in the BCO2 gene fed the control diet corresponded to the relatively lowest level of β-carotene and the relatively highest level of retinol in the liver. According to Lietz et al. [BCO1 activity can be affected by fats, proteins, antioxidants, carotenoids, and vitamin A status. This activity seems to be species-dependent. Xanthophyll supplementation at 20 mg/kg and 40 mg/kg had no effect on BCO1 mRNA expression in the liver of hens and chicks [In the control group, rabbits with the ins/del genotype in the z et al. , BCO1 acd chicks . It shouBCO2 gene were characterized by higher expression of the BCO2 gene than the animals with the ins/ins genotype. The observed phenomenon is probably dose-dependent because such a relationship was not noted in control group rabbits. The obtained results are complemented by the fact that rabbits with the deletion of both alleles, fed the marigold-supplemented diet, exhibited higher BCO2 gene expression than those receiving the control diet. As BCO2 del/del rabbits are able to absorb but not degrade xanthophylls, it is obvious that rabbits with this genotype belonging to the marigold group had the highest liver and serum lutein levels.In the marigold group, rabbits with the del/del genotype in the BCO2 genotype nor the diet affected the expression of genes associated with the metabolism of vitamins A and E , which is consistent with the absence of intergroup differences in the amounts of these vitamins in the liver. As reported by Gao et al. [LRAT expression in the liver of hens, but at the same time it increased the expression of another gene playing an important role in retinoid metabolism - STRA6 . This indicates the need for more comprehensive gene expression studies, including several genes involved in selected metabolic pathways.In the current study, neither the o et al. , xanthopBCO2 ins/ins, ins/del, and del/del rabbits, respectively, and they were higher than those obtained in the present study. However, the cited study involved rabbits raised until 140 days of age and fed a diet containing 37.70 mg/kg of lutein, whereas in the current experiment, rabbits were raised until 91 days of age and their basal diet had lower lutein content (21.64 mg/kg). In the control group, serum lutein levels were 1.80-fold and 1.48-fold higher in rabbits with the del/del genotype than in those with ins/ins and ins/del genotypes, respectively. Even more pronounced differences were noted in the marigold group where serum lutein levels were 2.96-fold and 2.91-fold lower in rabbits with ins/ins and ins/del genotypes than in del/del genotype animals, respectively. From the biochemical point of view, lutein exhibits substantial antioxidant activity in in vitro studies [E. coli, S. aureus, and P. aeruginosa, thus improving the health status of the host. Chew et al. [In the current study, lutein concentrations in the liver and serum were higher in del/del rabbits, compared with the other genotypes, regardless of the diet. In our previous research , hepatic studies , and it  studies . Kiplimow et al.  also desw et al.  demonstrw et al. ,30,43, wInterestingly, no intergroup differences were found in zeaxanthin concentrations in the liver of rabbits, which could result from the poor assimilation of xanthophylls . The assBCO2 gene, fed the marigold-supplemented diet, were characterized by the lowest level of LDH and the highest level of ALP in the blood plasma. In the literature, a decrease in LDH concentration is not considered an important finding [BCO2 alleles, including wild rabbits [Rabbits with the del/del genotype in the  finding , but an  finding . However finding . Moreove finding , and the rabbits ,30,51,52BCO2 del/del genotype contributes to increased accumulation of this micronutrient in the liver and blood of animals without compromising their health status or liver function. This information can be valuable for both rabbit breeders and rabbit meat consumers. Another implication of the study is that the BCO2 genotype may affect the expression levels of BCO1 and BCO2 genes in rabbits. Moreover, the addition of supplemental lutein to the basal diet of BCO2 del/del rabbits may increase the expression level of the BCO2 gene. These results may contribute to a better understanding of carotenoid metabolism in rabbits and, possibly, in other animal species. The present study has certain limitations, which may, however, pave the way for future research. Since this is the first study to determine changes in the expression of selected genes, depending on the BCO2 genotype of rabbits and the dietary inclusion levels of carotenoids, the obtained results should be confirmed in experiments involving the use of different diets, different rabbit breeds and, possibly, specialized cell lines. Moreover, gene expression was analyzed based on liver samples only, and only one gene from each metabolic pathway was examined. Therefore, the obtained data are fragmentary. Future research should focus on other tissues and organs, such as adipose tissue, eyes and the brain, and it should include more genes involved in the studied metabolic pathways.A novel finding of the present study is that an increase in the amount of lutein to 320 mg/kg in the diet of rabbits with the All experimental procedures were approved by the Local Ethics Committee in Olsztyn, Poland (consent No. 065.12.2020), and the animals were cared for in accordance with the guidelines of the European Parliament .n = 20, body weight of 726.1 g ± 66.09, mean ± SD) based on their genotype at codon 248 of the BCO2 gene . In this article, the gene variant with the AAT-deletion at codon 248 of the BCO2 gene is referred to as “del,” and the variant without the above deletion is referred to as “ins”. In order to determine the effect of the diet on carotenoid intake and/or storage, each experimental group was divided into two subgroups; one subgroup received a standard diet , and the other subgroup was fed a diet supplemented with 6 g/kg of Aztec marigold flower extract in the form of dried powder containing 4.96% of lutein and 0.28% of zeaxanthin . The ingredients and chemical composition of the control diet are presented in Sixty male Termond White growing rabbits raised in a closed facility on a farm in northern Poland were used in this study. Rabbits were divided into three experimental groups  until 91 days of age (August). Rabbits were housed individually under standard conditions, in wire-mesh flat-deck cages, which supported their visual contact with other rabbits. Temperature and relative air humidity were maintained within the range of 16–18 °C and 60–75%, respectively. The photoperiod was fully controlled, with 12 h of light and 12 h of darkness. The experimental facility had a forced ventilation system. At the end of the experiment, the animals were slaughtered by a veterinarian. They were bled within two minutes after physical stunning, and blood samples were collected into 2.5 mL heparinized test tubes. Immediately after skinning and evisceration, approximately 20 g samples of liver tissues were collected for chemical analyses. In order to determine RNA expression levels, liver samples were also snap frozen in liquid nitrogen immediately after slaughter, and were stored at −80 °C until RNA extraction.BCO2 gene was determined by PCR-RFLP, as described by Strychalski et al. [BCO2 intron 5 and exon 6, forward (5′GGGAATTCATCTGCATGGAC3′) and reverse (5′TCGATTCTGCAGGAGCAATA3′), were used for PCR. A total of 10 μL of the PCR product was digested with 10 U of AanI , and restriction fragments were separated by electrophoresis on 2.2% agarose gel with ethidium bromide.Genomic DNA was isolated from hair roots. Insertion/deletion polymorphism at codon 248 in the i et al. . BrieflyBCO1, BCO2, LRAT, and TTPA was determined by real-time PCR  with DyNAmo HS SYBR Green . GAPDH and β-actin were used as reference genes. About 2 μL of cDNA was used for each PCR reaction. The list of primers with sequences, annealing temperature, and amplification efficiency is presented in Approximately 100 µg of frozen liver samples were used for RNA isolation with the use of TRIzolTM Reagent , according to the manufacturer’s protocol. The concentrations of eluted RNA were measured with the NanoDrop 2000 spectrophotometer . The samples were stored at −80 °C for further analysis. Genomic DNA that remained in the samples after RNA isolation was digested with DNase I . Reverse transcription was carried out with the RevertAid First Strand cDNA Synthesis Kit , in accordance with the manufacturer’s instructions. The concentration of RNA for the synthesis of complementary DNA (cDNA) was standardized to 0.5 μg per sample. The expression of the gene encoding g, MPW-352R, MPW MED. Instruments, Warsaw, Poland) of heparinized blood samples, and it was stored at −70 °C until analyses. The following plasma biochemical parameters were determined on an automatic analyzer : the concentrations of total protein and albumin, and the activity of gamma-glutamyl transferase (GGT), asparagine aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP). The following antioxidant enzymes were determined : glutathione peroxidase (POX) and superoxide dismutase (SOD).Blood plasma was obtained by centrifugation  and extracted with ethanol and petroleum ether. The extracts  were rinsed with a 10% (w/v) aqueous solution of NaCl followed by distilled water, dehydrated with anhydrous sodium sulfate, and evaporated to dryness under a stream of nitrogen (N2). The residue was dissolved in 96% ethanol to determine the content of tocopherols and retinol, and in n-hexane to determine the content of β-carotene and xanthophylls.The concentrations of retinol and α-tocopherol in feed were determined by high-performance liquid chromatography (HPLC) in accordance with Polish Standards . Analytical samples were saponified  and saponified . Ethanol (35% v/v) and NaCl (10% w/v) were added to cooled samples, which were extracted twice with n-hexane (2 × 4 cm3) and centrifuged . Next, 3 cm3 and 4 cm3 samples of the supernatant were collected successively. The combined extracts were evaporated to dryness under a stream of nitrogen. The residue was dissolved in 1 cm3 of 96% ethanol to determine the content of tocopherols and retinol, and in n-hexane to determine the content of β-carotene and xanthophylls.The concentrations of retinol and α-tocopherol in animal tissues were determined as described by Hőgberg et al.  and Xu . One grav:v) as the mobile phase, a UV detector (326 nm for retinol) and a fluorescence detector , flow rate: 1 cm3 min−1, loop: 20 μl. The concentrations of retinol and tocopherol were estimated by comparing their peak areas with those of external standards -α-tocopherol–No Cat. T3251, retinol-vitamin A–alcohol-No Cat. R7632; the recoveries of α-tocopherol and retinol were 96% ± 2.8% and 89% ± 4.5%, respectively).The levels of retinol and α-tocopherol in feed, liver and blood serum were determined by reversed-phase (RP) HPLC , on a Nucleosil C18 column, with methanol/water  ; flow rate: 1 cm3 min−1, detector: UV-vis 450 nm; loop: 20 µL. External standards: β-carotene  and xanthophyll  standards from Sigma-Aldrich  [The hexane or petroleum ether extract of carotenoids (β-carotene and xanthophylls) was filtered through a 22 μm PTFE syringe filter (30-SF-02 CHROMACOL Ltd.) and analyzed by RP-HPLC , column: Gemini 5μm (Phenomenex), C18, 110 Å, 250 x4 mm; mobile phase: methanol:tetrahydrofuran (95:5 Germany) .All calculations were performed using R software . In ordeGAPDH and β-actin) were added as priors to the model function to account for variations in cDNA, and were allowed 1.2-fold changes [The results of qPCR were analyzed using the specialized MCMC.qpcr package  where a  changes . In charThe remaining data, i.e., the content of selected compounds in the liver and blood serum of rabbits, the values of blood plasma biochemical parameters, and serum antioxidant enzyme activities, were processed statistically by analysis of variance (ANOVA) followed by Duncan’s multiple range test. These data are presented in the tables as means ± standard deviations (SD).


Second harmonic generation microscopy (SHG) is generally acknowledged as a powerful tool for the label-free three-dimensional visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Despite the great need, progress in super-resolved SHG imaging lags behind the developments reported over the past years in fluorescence-based optical nanoscopy. In this work, we demonstrate super-resolved re-scan SHG, qualitatively and quantitatively showing on collagenous tissues the available resolution advantage over the diffraction limit. We introduce as well super-resolved re-scan two-photon excited fluorescence microscopy, an imaging modality not explored to date. Second harmonic generation microscopy(SHG) exploits the interaction of two incident near-infrared photons with a noncentrosymmetric molecule, resulting in a single emitted photon with double the energy via a nonlinear process involving virtual states. Considering its high imaging potential based on intrinsic contrast, augmented by other advantages such as no bleaching, no blinking, or high signal-to-noise ratio compared to fluorescence, SHG has gained important interest in the context of label-free characterization of fixed, ex vivo and in vivo tissues –3 and ad6) to reconstruct the improved resolution image, which has important implications with respect to the total acquisition time.Among the first notable attempts to achieve super-resolved images based on SHG, Masihzadeh et al.  and Liu Herein we introduce re-scan second harmonic generation microscopy (rSHG), a method for super-resolved SHG imaging, which contributes to overcoming the aforementioned limitations. We demonstrate as well re-scan two-photon excited fluorescence (rTPEF), with TPEF generally regarded as a complementary non-linear optical microscopy (NLO) technique to SHG . Both rSFor demonstrating the resolution advantage of rSHG we have used both a rat tail tissue fragment , a highly collagenous tissue that is emerging as a model for benchmarking SHG imaging methods, and also human breast tissue. In SI Appendix), with switching between the two work modes being done by modifying the angular amplitude of the scanner and re-scanner mirrors (SI Appendix). Noteworthy, the experimental FWHM value for the SHG image aligns to the theoretical value, which for 860-nm illumination and a 1.42 numerical aperture is 210 nm, according to In With the same setup we also explored rTPEF C, which The greater level of detail offered by rSHG over conventional SHG can obviously favor the more accurate extraction of quantitative information reflecting the morphology and other properties of the collagen fibrils , 17, whiIn terms of relevant applications, given that an RCM unit adapted for rSHG can be easily coupled to optical microscopes already available in clinical settings, we argue that histopathology laboratories can benefit from this reported methodology, as it can enable modern workflows based on NLO techniques to complement traditional assays. Such correlative approaches can be implemented on samples prepared using traditional histopathology protocols, as usual dyes  do not significantly affect the SHG signals. On the other hand, such stains have been shown to augment tissue imaging  with thiOverall, given the simplicity and flexibility of re-scan microscopy, we consider that the reported architecture and results are likely to augment the number and nature of applications relying on super-resolved non-linear optical imaging.SI Appendix. The tissue fragments of human origin discussed in this work were previously collected for routine histological examination from a patient and were deidentified prior to use in the current study.rSHG and rTPEF were performed with a multimodal prototype based on a conventional RCM setup of first generation that was custom-modified. The architecture and configuration of this system, together with aspects on data acquisition and sample preparation, are presented in Supplementary File


Fanconi anemia (FA) is a rare disease of bone marrow failure. FA patients are prone to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the molecular clonal evolution of the progression from FA to MDS/AML remains elusive.Herein, we performed a comprehensive genomic analysis using an FA patient (P1001) sample that transformed to MDS and subsequently AML, together with other three FA patient samples at the MDS stage.UBASH3A, SF3B1, RUNX1 and ASXL1 gradually appeared at the later stage of MDS, while the IDH2 alteration become the dominant clone at the leukemia stage. Moreover, single-cell sequencing analyses further demonstrated a polyclonal pattern was present at either MDS or AML stages, whereas IDH2 mutated cell clones appeared only at the leukemia stage.Our finding showed the existence of polyclonal pattern in these cases at MDS stage. The clonal evolution analysis of FA case (P1001) showed the mutations of We thus propose a clonal evolution model from FA to MDS and AML for this patient. The results of our study on the clonal evolution and mutated genes of the progression of FA to AML are conducive to understanding the progression of the disease that still perplexes us.The online version contains supplementary material available at 10.1186/s40164-022-00319-5. Fanconi anemia  is a rare autosomal recessive, X-linked (FANCB) or autosomal dominant (FANCR/RAD51) bone marrow failure disease, which occurs at a rate of 1–5 cases per million –4. TheseClinically, FA is mainly characterized by developmental abnormalities, bone marrow failure (BMF), and predisposition to cancer . BMF is Distinct clone formation is an evolutionary ability that offers both benefits and drawbacks. Moreover, the clonal evolution has long been implicated as a risk factor for the development of many diseases, including MDS and other cancers , 10. Stu+CD19+), T cells (CD45+CD3+), NK cells (CD45+CD16/56+), Monocytes (CD45+CD14+) and Granulocytes (CD45+CD10/33+CD14−) in FA stage, and from PB cells during MDS and AML stage. Genomic DNA extracted from folliculus pili samples were used as germline control for somatic variant calling. For patient P1002, P1003, and P1004, genomic DNA were extracted from whole bone marrow cells, and folliculus pili samples at MDS Stage. All Genomic DNA of whole BM, whole PB, BMMNCs, PBMNCs, sorted cells, and folliculus pili samples were extracted by DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer’s protocol. All patients provided written consent on a protocol approved by the Clinical Research Ethics Committee of Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences (HG2021007-EC-1).Peripheral blood (PB), bone marrow (BM), and folliculus pili samples were obtained from the patient P1001. Bone marrow and folliculus pili samples of the other three patients who converted from FA to MDS were derived from P1002, P1003, and P1004. For patient P1001, genomic DNA was extracted from MDS and AML bone marrow mononuclear cells, as well as from 5 types of blood cells  following manufacturer’s instructions. PBMNCs at the first diagnosis of patient P1001 were stained for 30 min on ice with antibodies: CD45, CD3, CD19, CD16/56, CD14, CD10/33 to isolate T cells (CD45+CD3+), B cells (CD45+CD19+), NK cells (CD45+CD16/56+), Monocytes (CD45+CD14+), Granulocytes (CD45+CD10/33+CD14−). Then cells were sorted on an Aria III flow cytometer. For single-cell sorting, we used Lin−CD34+CD38− to enrich for hematopoietic stem cells. After staining of surface markers, single cells were directly sorted into a 96-well PCR plate using Aria III flow cytometer. A list of antibodies is provided in Additional file Whole BM and peripheral blood samples were obtained from patients during the stage of FA. Mononuclear cells (MNCs) of bone marrow and peripheral blood were enriched by Ficoll  samples. Paired-end sequencing was performed using the Illumina HiSeq platform with 150 bp read length. For all samples, 65 to 100 Gb of WGS sequence was generated, corresponding to 40 × to 50 × haploid coverage of the reference genome.® Single Cell WGA Kit (Vazyme) according to the manufacturer’s protocol, which uses a multiple displacement amplification (MDA)-based isothermal amplification system to achieve indiscriminate amplification of the whole genome using a single cell or micro-sample as a template [Single cell-genome amplification was performed with Discover-sctemplate . Then prAfter single cell-genome amplification, targeted sequencing was performed. For analysis of single-cell targeted sequencing data, we removed low quality reads by Trim Galore v0.4.4 using the default parameters. Therefore, the quality reads were mapped to hg38 with BWA v0.7.15  and thenThe clinical features of 4 patients that participated in this study are summarized in Table To analyze clonal evolution during the progression with FA-AA to MDS and AML in patient P1001, 4 peripheral blood samples of three stages , 3 bone marrow samples of MDS and AML stages , and folliculus pili samples (as control samples) from the P1001 were obtained and subjected to whole-genome sequencing  emerged. The ASXL1 is a member of the Polycomb group of proteins, and mutations in ASXL1 were shown to be associated with myelodysplastic syndromes and chronic myelomonocytic leukemia [IDH2 is isocitrate dehydrogenase 2 and its mutations were reported to be associated with the pathogenesis of AML [To prospectively analyze clonal evolution during the progression of FA-AA to AML, we inferred the clonal architectures based on the VAFs of mutations. Five clonal groups were identified based on clusters of VAFs Fig. d, e. Thes of MDS . When thleukemia . IDH2 iss of AML . Based oASXL1, ERBB4, MCFD2, MCM10, POTEH, SF3B1 and WLS were shared across all stages of the progression from MDS to AML  and WNT7A as AML-specific mutations, respectively  began to emerge, and the CCF of R140W mutant is high in both PB and BM samples, suggesting that mutation of IDH2 may play a key role in the transition from MDS to AML. Meanwhile, SPG7 and ELF4 were specific mutations in whole bone marrow cells and peripheral blood cells, respectively  were found to be enriched for MDS-SC (MDS-stem cell) or AML-SC [To explore the relationship between somatic mutations and cell surface markers of leukemia stem cells, we combined single-cell targeted sequencing with FACS data. We found that the fraction of Linr AML-SC . We thusStudy has shown that genomic instability of patients with Fanconi anemia, gain or loss of large fragments of chromosomes may affect the progression of the disease . To clarCytogenetic abnormalities are frequently found in Fanconi anemia . To furtFANCA, FANCD2 and FANCL, respectively were used for whole genome sequencing. Consistent with patient P1001, an elevated percentage of C > T/G > A was found and is consistent with molecular change signatures in AML (Fig. IDH1 might drive the progression of disease with its higher VAF, while FCGBP and ADAMTS7 could expedite FA transformation to MDS for patient P1003 (Fig. To determine the mutation burden and evolution process of FA patients that carry different mutant FA genes, we next examined the somatic mutation burdens of FA-MDS patients. BM samples from three different FA-MDS patients with mutations of AML Fig. b. HoweveAML Fig. c. FurtheAML Fig. d. For pa003 Fig. d. Taken UBASH3A, SF3B1, RUNX1 and ASXL1 mutations gradually appeared and became the major clones at MDS stage, while IDH2 gradually emerged as the dominant clone at leukemia stage. Additionally, our finding also showed the distinct bone marrow karyotypes and genetic alterations at MDS and AML. C > T and G > A transitions were the most abundant SNVs, which was in line with previous reports for AML cases [ASXL1 were detected at stem cell level at the early stage of MDS in the bone marrow, but appeared later in the peripheral blood in this patient. This may either due to that these mutations have lower VAF in PB than in BM at the current sequencing depths [The molecular mechanism regarding the clonal evolution from AA to MDS and AML in FA patients remains elusive. Herein, a novel clonal evolution route was identified in patient P1001 with polyclonal pattern at both MDS and AML stages based on whole genome and single-cell sequencing analysis. The ML cases . Interesg depths , 43, or g depths .ASXL1 mutation was persistently detected throughout the disease progression although the mutation including clone appeared more apparent at late state of MDS. Interestingly, the clonal cells carrying FBH1 and HUWE1 mutations appeared at the later stage of MDS, whereas UBASH3A, FBH1 and HUWE1 mutations disappeared at AML stage. ASXL1 is an epigenetic modulator and its mutation is frequently related to clonal hematopoiesis, MDS and AML [ASXL1 mutations were found to be are mutually exclusive to DNMT3A, FLT3-ITD, NPM1 or SF3B1 mutations. However, in our study, the FA patient carrying ASXL1 mutation coexisted with the mutations of IDH2 and SF3B1, probably reflected a unique character of FA patient due to impaired DNA repair pathway [IDH2 mutation was established, the clone containing IDH2 mutation proliferated and prevailed in AML. Our single-cell sequencing data suggested continuous competition and disappearance of distinct clones during AML progression at MDS stage, which was supported by the sequencing data of other three MDS patients, showing highly heterogeneous mutations at the MDS stage. It is the mutation of genes related to disease progression acquired by clonal cells that determines how fast the progression of the disease.In our study, a clone with  and AML –46. In A pathway . FBH1 he pathway , 49. HUW pathway . At the UBASH3A, SF3B1, RUNX1, ERBB4 and MCM10 are associated with NF-κB, Ras-MAPK-ERK, P13K-Akt and Wnt/β-catenin pathway abnormalities [IDH2 was the primary clone of AML, and the mutation of IDH2 could be considered as the third hit of disease progression in patient P1001.Cells from FA patient may undergo multiple hits during the progression of FA in addition to their FA gene mutation (modeled in Fig. malities –59. ActiIDH2 was shown to be associated with an improved prognosis in intermediate-risk AML patients [IDH2 mutations are closely linked to the mutations of DNMT3A, SRSF2, NPM1, ASXL1 and RUNX1. Old adults with newly diagnosed IDH2-mutant AML benefited from Enasidenib, an inhibitor of IDH2-mutant protein [Notably, patients . Further protein . Thus, IIn conclusion, we present a novel and dynamic model for the clonal evolution of FA disease progression using single cell sequencing with sequential samples from a single patient. With this model, we can determine which mutations are responsible for cells acquiring tumor forming characteristics when the disease progresses to AML. Moreover, our model provides insights into a molecular basis and treatment strategy for FA disease. With the ongoing expansion of sample size, our approach may identify more potential molecular targets for effective clinical treatment of FA and its progression.Additional file 1: Table S1. The mutation results in bone marrow and peripheral blood samples from patient P1001 at different stages. Table S2. Primers for single-cell targeted sequencing. Table S3. Antibodies for FACS experiments. Material methods. Including Whole genome analysis, Clonal analysis, and Single-cell targeted sequencing.