Family-Based Treatment (FBT) is the first line of care in paediatric treatment while adult programs focus on individualized models of care. Transition age youth (TAY) with Anorexia Nervosa (AN) are in a unique life stage and between systems of care. As such, they and their caregivers may benefit from specialized, developmentally tailored models of treatment.The primary purpose of this study was to assess if parental self-efficacy and caregiver accommodation changed in caregivers during the course of FBT-TAY for AN. The secondary aim was to determine if changes in parental self-efficacy and caregiver accommodation contributed to improvements in eating disorder behaviour and weight restoration in the transition age youth with AN. Twenty-six participants (ages 16–22) and 39 caregivers were recruited. Caregivers completed the Parents versus Anorexia Scale and Accommodation and Enabling Scale for Eating Disorders at baseline, end-of-treatment (EOT), and 3 months follow-up.p = .398). Parental self-efficacy significantly increased from baseline to 3 months post-treatment (p = .002). Caregiver accommodation towards the illness significantly decreased from baseline to EOT (p = 0.0001), but not from baseline to 3 months post-treatment (p = 1.000). Stepwise ordinary least squares regression estimates of eating disorder behaviour and weight restoration did not show that changes in parental-self efficacy and caregiver accommodation predict eating disorder behaviour or weight restoration at EOT or 3 months post-treatment.Unbalanced repeated measures designs for parental self-efficacy and caregiver accommodation towards illness behaviours were conducted using generalized estimation equations. Parental self-efficacy increased from baseline to EOT, although not significantly (Our findings demonstrate, albeit preliminary at this stage, that FBT-TAY promotes positive increases in parental self-efficacy and assists caregivers in decreasing their accommodation to illness behaviours for transition age youth with AN. However, changes in the parental factors did not influence changes in eating and weight in the transition age youth. Family-Based Treatment for Transition Age Youth were delivered to 26 participants with Anorexia Nervosa (ages 16–22) and their families. This study evaluated how family members responded to eating disorder (ED) behaviours throughout the course of this treatment. This study also evaluated how confident family members felt about their ability to help their loved one with ED behaviours throughout the course of treatment. The impact of changes in parental responses and confidence in helping with eating disorder symptoms and weight gain was examined. Throughout the course of treatment caregiver accommodation to eating disorder behaviour decreased and feelings of parental self-efficacy increased. However, changes in parental self-efficacy and accommodation did not predict changes in eating disorder behaviour and weight restoration in transition age youth with AN.Anorexia Nervosa (AN) is a life threatening mental health condition  with sevThere are a variety of family therapy approaches developed for the treatment of AN such as Family Therapies for AN (FT-AN) and Multi-Family Therapy for AN (MFT-AN) . The curAlthought studies of FBT indicate that it is an effective treatment  and thatTransition age youth experience many transitions that define their developmental stage, such as post-secondary education, increased fiscal responsibility, changing geographic location, and increased personal responsibility and autonomy . For traAn open trial of FBT for transition age youth (FBT-TAY) that encouraged negotiation between supportive caregivers and the independence of the youth has recently shown promising outcomes for individuals with AN . FBT-TAYParental self-efficacy has been identified as a potential mechanism of change that promotes positive treatment outcome in FBT , 25. ParCaregivers may also engage in behaviours that inadvertently exacerbate ED symptoms , 28. EatThe primary aim of the study was to determine if parental self-efficacy and caregiver accommodation changed in a course of FBT-TAY from baseline to the end-of-treatment (EOT), and 3 months post-treatment. The second aim of the study was to determine if changes in parental self-efficacy and caregiver accommodation influenced changes in eating disorder behaviour and weight restoration at EOT and 3 months post-treatment in transition age youth with AN.An open trial of FBT-TAY was conducted across one adult and two pediatric hospital sites in Ontario, Canada between August 2014 and September 2016. The acceptability, feasibility, and impact of FBT-TAY on eating disorder behaviors and weight restoration is described by Dimitropoulos et al. . Three hAll potential participants were offered treatment as usual (TAU) or FBT-TAY. FBT was the TAU in both pediatric hospital sites (ages 12–18). In the pediatric hospitals, potential participants were offered either traditional FBT or FBT-TAY. At the adult hospital site, TAU was an intensive cognitive behavioral therapy (CBT) based group program within an inpatient (average stay of 5 months) or day hospital (average stay of 5 weeks) setting. In the adult hospital, family therapy was an optional component of treatment. At the point of assessment, if a potential participant met eligibility criteria (described below) for the FBT-TAY open trial, they were referred to the study coordinator. Potential participants were asked to identify at least one caregiver to participate in treatment. To support the autonomy of the transition age youth, caregiver selection was entirely left to their discretion and was defined broadly to include parents, guardians, siblings, partners, extended family and/or friends. All caregivers and participants with an eating disorder gave informed consent and were then assigned to a FBT-TAY study therapist. Further details about the methodology and participants are described by Dimitropolous et al. .FBT-TAY includes 25 sessions over the course of three phases . These pM = 18.15 years, SD = 2.11). The majority of participants were female (96.2%), Caucasian (61.5%), single (88.5%), living with family or relatives (92.3%), unemployed (53.9%), and had completed some high school education (46.2%). A total of 39 caregivers   were included in the analysis. Two parents did not complete the questionnaires required for inclusion in the analysis. The majority of caregivers were Caucasian (66.7%), married/partnered (79.5%), employed full-time (74.4%), and had an undergraduate university degree or higher (61.5%). See Table A total of 26 young people participated in this study  is a 33-item, self-report measure which comprises four subscales  . Each su2) according to age and sex [For each participant aged 16–20, the weight restoration goal was comprised their median BMI (kg/m and sex . For eacThe Parents versus Anorexia (PvA) scale was developed to assess parental self-efficacy in the role of re-nourishing a child back to health . This inThe Accommodation and Enabling Scale for Eating Disorders (AESED) is a 33-item self-report scale which is used to assess the degree to which caregivers accommodate and enable illness behaviours in their loved one . ResponsMissing data was addressed using multiple imputation  The procp values are reported for each GEE that was performed.Analyses were performed in IBM SPSS Statistics Version 24. The primary objective of this study was to investigate changes in parental self-efficacy and caregiver accommodation from baseline to end of treatment and from baseline to 3 months post-treatment. Due to the unbalanced and correlated nature of the data, unbalanced repeated measures designs for parental self-efficacy and caregiver accommodation were conducted using generalized estimation equations (GEEs) to determine the time effect while controlling for each participant. Separate analyses were undertaken for each of the predictors  due to concerns with multicollinearity. Bonferroni adjusted The second objective of this study was to investigate the impact of parental self-efficacy and caregiver accommodation to the illness on eating disorder behaviour and weight restoration in the transition age youth with AN. Ordinary Least Square (OLS) regression estimates of eating disorder behaviour and weight restoration were conducted at end of treatment and 3 months post-treatment. Separate regression analyses were undertaken for each of the predictors (changes in PvA and changes in AESED) due to concerns with multicollinearity. First, we examined whether the changes in parental self-efficacy from baseline to end of treatment predicted eating disorder behaviour and weight restoration at the end of treatment. Second, we examined whether the changes in parental self-efficacy from baseline to 3 months post-treatment predicted eating disorder behaviour and weight restoration at 3 months post-treatment. Third, we examined whether changes in caregiver accommodation from baseline to end of treatment predicted eating disorder behaviour and weight restoration at end of treatment. Finally, we examined whether changes in caregiver accommodation from baseline to 3 months post-treatment predicted t eating disorder behaviour and weight restoration at 3 months post-treatment. Z-scores were created for each variable in order to standardize measurement. This process is imperative when performing OLS regressions from multiply imputed data .2 = 11.95, p = .003). Pairwise comparisons revealed that the total mean score of parental self-efficacy did not significantly increase from baseline  to EOT , but did increase significantly increased from baseline  to 3 months post-treatment .Table 2 = 37.45, p = .0001). Pairwise comparisons revealed that the total mean score of caregiver accommodation significantly decreased from baseline  to EOT , but not from baseline  to 3 months post-treatment .Table Table Table The current study of FBT for transition age youth with AN aimed to explore how parental self-efficacy and caregiver accommodation to the illness changed over the course of treatment. We further sought to identify whether such changes in parental self-efficacy and caregiver accommodation were predictive of changes in eating disorder behaviour and weight in the transition age youth at EOT and/or three-months post-treatment. We found that parental self-efficacy increased, but not significantly, from baseline to EOT. However, parental self-efficacy increased significantly from baseline to 3 months post-treatment. The results further revealed that accommodation to the illness decreased significantly from baseline to EOT, but not from baseline to 3 months post-treatment. Neither parental self-efficacy nor caregiver accommodation predicted change in ED symptoms and weight in the transition age youth at EOT or 3 months post-treatment. Overall, this study demonstrated that caregivers became increasingly more confident in their ability to support their loved one with AN, and perceived themselves as engaging less with/or permitting fewer ED symptoms throughout treatment. However, these changes did not predict change in the transition age youth’s eating disorder symptoms or weight.Transition age youth are between pediatric and adult systems of care, which requires special treatment attention to their unique developmental challenges. The experience of both wanting the support of family and friends while also negotiating boundaries for increased autonomy and independence can complicate how caregivers and transition age youth work together to diminish AN behaviours. Previous research on adolescent FBT found decreased parental self-efficacy and fear was linked with higher accommodation to the illness . When faIt is important to note that the tool used to assess parental self-efficacy, the PvA, was developed for and is primarily used to assess empowerment in the context of child and adolescent treatment of EDs using FT . Items oInterestingly, despite the limitations of the scale and the collaborative focus of FBT for TAY, caregivers still experienced a significant increase in parental self-efficacy from baseline to EOT. Working to empower parents and reduce fear and self-blame is a focus of FBT and other family therapies for AN (ex: FT-AN) . TherefoThe collaborative approach outlined above may be counter intuitive to families at this life stage where it is developmentally appropriate for young adults and parents to begin separating financially, geographically, and shifting their relationship from one of dependence to interdependence , 22. HowThe result of increased parental self-efficacy over the course of FBT-TAY provides context for the caregiver’s significant decrease in accommodation behaviours over the course of treatment. Despite the unique features of transition age youth, caregivers in our sample were comparable to other adolescent and adult caregiver samples in terms of baseline accommodation and enabling scores –44. The In the current study, neither parental self-efficacy nor accommodation predicted change in eating disorder symptoms. This differs from past literature in the treatment of adolescent AN has found that parental self-efficacy increases during treatment with FBT . In a stFBT-TAY differs from standard manualized FBT . It begiTherapies that swiftly target caregiver beliefs about their ability to support a loved one are very important in facilitating their efforts to address disordered eating particularly during meal times. A caregiver’s ability to cope and provide support is impacted over time by feelings of fear and self-blame which erode a caregiver’s efficacy  , 47, 48.It is possible that FBT-TAY can be effective in halting or reversing caregiver disempowerment as well as decreasing accommodation behaviours in caregivers during the course of treatment. This is an important advancement in terms of our knowledge of the role of caregivers in the treatment of EDs in transition age youth. Currently, care for transition age youth receiving treatment in adult systems do not routinely involve caregivers, but instead prioritize individual or group-based models. Therefore, caregivers who are still intimately involved in the daily life of transition age youth are typically excluded from treatment of those over the age of 18. This is a problematic systemic issue that the current study proposes can be amended by introducing a developmentally appropriate model of treatment that recognizes the role that parents can play in supporting their adult child while also empowering the individual with the illness. Programs should begin to identify the unique needs of transition age youth and their caregivers to better serve this population. Future research should establish the efficacy of FBT-TAY via a randomized controlled trial. We also recommend furture research evaluating FBT-TAY to other family based treatment modalities like FT-AN or MFT-AN .FBT-TAY was manualized and all study therapists were provided initial training followed by weekly supervision throughout the study. Even though the study used a novel model of treatment, it was developed using direct feedback from clinicians focused on working with transition age youth with AN . FinallyThe current study is the first to describe the impact of FBT-TAY on caregivers of transition age youth with AN Acknowledging the unique needs of transition age youth who are between pediatric and adult systems of care is an important line of inquiry given the limited involvement of family members in treatment for older adolescents and young adults. The current study aimed to identify if involving family in the treatment of transition age youth using FBT-TAY would elicit benefits in the caregivers. This study demonstrates that FBT-TAY has promise and may lead to changes in caregivers of transition age youth with AN presenting for treatment in both pediatric and adult programs. Future studies should assess the impact of treatments on caregivers given the mental health impacts  of caregiving , and the positive treatment impacts for those with an ED .


The magnetic domain patterns of amorphous bilayered FeSiB/FeNbSiB and FeNbCuSiB/CoSiB ribbons are observed and analysed using the magneto-optical Kerr microscopy (MOKM) and magnetic force microscopy (MFM). Both microscopic techniques are highly sensitive to the sample surface; possibility of Kerr microscopy to visualize the domains separately in both layers is achieved by focusing the laser spot on the ribbon cross section. Wide curved domains as well as fine fingerprint domains were detected at the surface of ribbons due to presence of local stresses coming from the preparation process. With respect to high lateral resolution of MFM and its out-of-plane magnetization sensitivity, the perpendicularly magnetized crossed stripe domain patterns can be selected as well. Coiling of the ribbons on the half-round-end sample holder is often used to induce and control the magnetic anisotropy of these alloys. Changes in the magnetic domain structure at the outer-coiled surface and its dependence on the sign of magnetostriction coefficient are discussed in detail. Finally, the MFM images in the presence of external in-plane magnetic field up to ±40 kA/m are shown. The amorphous and/or nanocrystalline alloys are deeply examined by many research teams world-wide due to their excellent soft magnetic properties , 2. TheyRecently, the innovations of PFC technology connected with the integration of double-nozzle allow the preparation of bilayered (BL) and/or multilayered (ML) functional materials. They are used mainly in sensor applications, like deflection sensors  and dispNowadays, the magnetic force microscopy (MFM) is advanced well established surface-sensitive technique for magnetic domain observations in a variety of magnetic materials  and Fe73.5Nb3Cu1Si13.5B9/Co72.5Si12.5B15 (BL-FC) ribbons were prepared by PFC technique using crucible divided into two chambers [As-cast, 36 Hs necessary to saturate the sample and the surface image is stored as a reference. Then the value of magnetic field is gradually decreased and we observe the difference between the actual image at applied magnetic field H < Hs and reference. In most cases the MOKM domains were investigated in remnant state, that is, after switching off the magnetic field (H = 0). Sensitivity to individual magnetization components can be adjusted using the aperture diaphragm. Opening and closing of the diaphragm enable illuminating different areas of the conoscopic image, “Maltese cross,” occurring in the microscope back focal plane. As seen in Magnetic domains were investigated by two surface-sensitive techniques. Magneto-optical Kerr microscopy (MOKM) consists of specially designed polarization microscope for direct observation of magnetic domains; see schematic description in MOKM magnetic domain investigation was supplemented by atomic/magnetic force microscopy (AFM/MFM) measurements with and/or without external magnetic field. The AFM/MFM experiments were carried out in air at room temperature with the scanning probe microscopy (SPM) platform  using the Co-Cr coated cantilevers see  in semicThe AFM/MFM setup takes advantage from longitudinal magnetic field generator (electromagnetic coil) that is able to create the magnetic field along the sample surface up to 80 kA/m see . To suppThe surface domains of BL-FF and BL-FC samples measured by MOKM are given in Generally we can say that each ribbon place will have its own unique magnetic domain structure reflecting on the one hand stresses coming from preparation process and on the other hand postpreparation treatment. Differences between the ribbons and their sides are visible from λs in both layers of BL-FF ribbon induces the easy magnetization axis in the ribbon axis at both surfaces.The magnetic behaviour observed at the ribbon interface using the MOKM is shown in Due to higher roughness of the ribbon wheel sides see  only theFigures However, there are also places, where the crossed stripe domains completely vanish. Those regions can be found on the surfaces of both ribbons. An example of such situation is depicted in the case of BL-FC sample . We expeHext. Results are shown in Hext is indicated by the arrows. The bottom figure is divided into two parts each corresponding to the positive and negative values of applied field during the same measurement, respectively, where upper and lower row of images refer to the domain structure obtained for positive and negative values of Hext = 4, 8, 16, and 40 kA/m. Because both the sample and the tip are exposed to the in-plane external magnetic field, their out-of-plane magnetization components become weaker at the expense of increasing the in-plane ones. This influences the magnetic interaction between the tip and the sample and stripe domain patterns differ from that observed without applied Hext. One can see slow broadening of the stripes with increasing Hext. At 16 kA/m the sample is partly saturated and the magnetic domain structure completely disappeared after reaching 40 kA/m. Then the external magnetic field was switched off (remnant state) and its amplitude was further increased with the opposite polarity. However, the remnant magnetization of the sample from the previous step is responsible for changes in the domain patterns in comparison to the positive magnetic field polarity. Therefore, stripe domain arrangement is visualized even at 40 kA/m showing practically symmetric zig-zag domain pattern.In Combination of magneto-optical Kerr microscopy (MOKM) and magnetic force microscopy (MFM) was successfully used for observations of magnetic domain patterns at surfaces and at cross section of bilayered FeSiB/FeNbSiB and FeNbCuSiB/CoSiB ribbons. The following types of domains have been detected and discussed.Fingerprint Domains. Surface closure domains indicating the presence of out-of-plane magnetic anisotropy in the ribbon bulk, coming from local planar compressive stresses originated during the ribbon preparation. MOKM shows high surface magnetic contrast arising from strong in-plane magnetization component and weak out-of-plane magnetization component; MFM exhibits lower magnetic contrast due to the sensitivity to weak out-of-plane magnetization component. MFM response completely vanishes in external magnetic field of 40 kA/m due to the ribbon saturation.Wide Curved Domains. In-plane magnetic domains coming from local tensile stresses, their directions reflect random orientation of local in-plane easy magnetization axis on the surface. MOKM detects wide curved domains at the outer tensile-stressed side thanks to the sensitivity to in-plane magnetization component; practically no corresponding MFM response was observed.


Medicago sativa. Light-sheet fluorescence microscopy (LSFM) is an advanced imaging technique widely used in animal developmental biology, providing efficient fast optical sectioning under physiological conditions with considerably reduced phototoxicity and photobleaching. Long-term 4D imaging of living plants offers advantages for developmental cell biology not available in other microscopy approaches. Recently, LSFM was implemented in plant developmental biology studies, however, it is largely restricted to the model plant Arabidopsis thaliana. Cellular and subcellular events in crop species and robust plant samples have not been studied by this method yet. Therefore we performed LSFM long-term live imaging of growing root tips of transgenic alfalfa plants expressing the fluorescent molecular marker for the microtubule-binding domain (GFP-MBD), in order to study dynamic patterns of microtubule arrays during mitotic cell division. Quantitative evaluations of cell division progress in the two root tissues (epidermis and cortex) clearly indicate that root growth rate is correlated with duration of cell division in alfalfa roots. Our results favor non-invasive environmental LSFM as one of the most suitable methods for qualitative and quantitative cellular and developmental imaging of living transgenic legume crops.Cell division and expansion are two fundamental biological processes supporting indeterminate root growth and development of plants. Quantitative evaluations of cell divisions related to root growth analyses have been performed in several model crop and non-crop plant species, but not in important legume plant  Plant growth and development is directed by key biological processes establishing proper morphogenetic pattern. Among them, cell division plays an indispensable role. Cell divisions in the root meristem are followed by subsequent cellular expansion in the root elongation zone and ensure sustained development of roots. Plant mitotic cell division is dynamic process controlled by rearrangements of microtubules that are gradually transformed into different microtubule arrays during mitosis and cytokinesis. Before the onset of cell division, cortical microtubules are narrowing to form the preprophase band (PPB) defining the cell division plane  and spinning disk (SD) microscopy are most popular ones. From practical point of view, dimensions of tobacco BY-2 and Arabidopsis suspension cells, as well as easily cultured Arabidopsis seedling plants facilitate utilization of conventional microscopy methods for live-cell imaging of mitotic microtubules in dividing cells. Another important aspect of microscopic cell division studies on model plants is availability of firmly established transformation protocols and protein tagging approaches in Arabidopsis and tobacco. Some of these studies analyzed stem cell divisions  and suffer from high energy illumination causing undesirable phototoxicity and photodamage. In comparison to Arabidopsis, crop species including alfalfa typically have more robust habitus and their roots are thicker because they consist from more tissue layers. Consequently, mounting of the whole alfalfa seedlings to classical horizontally-positioned microscopy stage is more challenging and affects fitness of such samples during long-term experiments. Obviously, microscopic techniques like CLSM and SD also suffer from serious limitations in imaging depth. Finally, all commercial CLSM and SD platforms are based on horizontal stages, not allowing for living plants to be observed normally oriented with respect to the gravity vector. All these disadvantages can be avoided by using light-sheet fluorescence microscopy (LSFM). Optical sectioning of the sample with excitation sheet of light is very fast. Importantly, excitation of fluorophores is restricted within the light-sheet volume, which effectively eliminates out-of-focus fluorescence and bleaching. Emitted fluorescence light is detected by independent orthogonally-positioned detection objective. Focal plane of the light-sheet is harmonized with the focal plane of the detection objective and thus, very thin layer of the sample is detected by spherical aberration-free imaging. Sample can be freely rotated with respect to the light-sheet plane illumination allowing multi-angular image acquisition. Fast imaging mode of LSFM ensures that imaged cells are illuminated with very low level of excitation energy Stelzer, . FinallyM. sativa are more difficult due to the limited availability of established molecular tools and efficient transformation techniques. In contrast to Arabidopsis and tobacco, there are only scarce reports on cytoskeletal dynamics during cell division in Medicago sativa. It is known that transitions between G1/S and G2/M phases are controlled by cyclin-dependent Ser/Thr kinases, among others also by M. sativa kinase CDKB2;1  of the mammalian MICROTUBULE-ASSOCIATED PROTEIN 4 (MAP4) fused to GFP driven by constitutive 35S promoter. N-terminal GFP fusion with rifampicin and kanamycin resistance was prepared by classical cloning method in pCB302 vector with herbicide phosphinothricin as the selection marker in planta. Phosphinothricin has been added to each subculture medium to control successful transformation. The transformation procedure was performed according to protocol for efficient transformation of alfalfa described by Samac and Austin-Phillips  for appropriate time in the culture chamber at 22°C, 50% humidity and 16/8 h light/dark photoperiod  or into Murashige and Skoog plant maintaining medium (MS). Embryos were enclosed from the upper part by fluorinated ethylene propylene (FEP) tube with an inner diameter of 4.2 mm and wall thickness of 0.2 mm . FEP tubes were inserted into the culture medium carefully to enclose individual somatic embryo and surrounding medium inside of the tube. After 1–2 days of somatic embryo stabilization and after visual inspection of the root pole which was starting elongation, such FEP tube with somatic embryo was carefully removed from culture medium and prepared for light-sheet microscopy. Sample was prepared according to the “open system” protocol for long-term live-cell imaging of Arabidopsis thaliana seedlings described by Ovečka et al. , which was cut-open at the tip before use. Plastic syringe was used for fixing the sample in sample holder of the microscope. Sample holder with the sample was placed into observation chamber of the microscope tempered to 22°C using Peltier heating/cooling system. Before insertion of the sample to the microscope somatic embryo in the solidified culture medium was ejected slightly out of the FEP tube to the extent that root in the block of solidified culture medium was directly loaded into appropriate liquid medium (MMS or MS) in the microscope chamber. FEP tube was still holding the sample, but was no more surrounding the root part of the plant, which enhanced considerably quality of root deep imaging. To prevent contamination during long-term imaging, liquid medium filling the observation chamber was filter-sterilized using a sterile syringe filter. FEP tube was sterilized using 70% ethanol and dried out before use. After insertion of the sample to the observation chamber of the light-sheet microscope and 30 min stabilization, it was prepared for imaging.Transgenic somatic embryos carrying a et al. , with a Long-term fluorescence live-cell imaging was done with the light-sheet Z.1 system , equipped with W Plan-Apochromat 20×/1.0 NA water immersion detection objective  and two LSFM 10x/0.2 NA illumination objectives . Samples were imaged using dual-side illumination by a light-sheet modulated with a pivot scan mode. GFP-MBD was visualized with excitation line 488 nm and with emission filter BP505-545. Laser excitation intensity did not exceed 3% of the laser intensity range available. Image acquisition was done every 5 min in Z-stack mode for a period of 3–12 h. Scaling of images in x, y, and z dimensions was 0.152 × 0.152 × 0.469 μm. Images were scanned in two or three views coordinated to follow each other in y axis to record root growth in long-term imaging and to prevent the movement of the growing root out of the field of only one field of view. Images were recorded with the PCO.Edge camera  with the exposure time 20 or 50 ms per optical section.Time-lapsed images of growing roots in x-, y-, z-, and t-dimensions were acquired using Zen 2014 software, black edition . Images in the czi format were transformed to 2D images using Maximum intensity projection function. Subsets of data containing individual dividing cells were created either with defined x- and y- dimensions from maximum intensity projection images or from original data with defined x-, y-, and z- dimensions. Root growth rate and displacement of individual cells in growing roots  according to one-way ANOVA and subsequent Fisher's LSD test at the level of significance of M. sativa plants originating from somatic embryos. After completion of somatic embryogenesis and after maturation, further development of somatic embryos required “germination,” characterized by proliferation on the root pole. This was initiated by transfer of mature somatic embryos from a hormone-free to a root and plant development (MMS) inducing medium. Somatic embryos with already proliferating roots were transferred directly to plant maintaining (MS) medium. Each somatic embryo has been transferred to MMS (or MS) medium individually in such way so that the root pole of somatic embryo was carefully inserted vertically into the solidified culture medium. This ensured that further growth of the root would continue inside of the medium and in vertical orientation. Upon activation of root growth as defined by its visible elongation, such “germinating” somatic embryos were prepared for light-sheet microscopy using FEP tubes.Light-sheet microscopy imaging was performed on roots of transgenic −1 were characterized as fast-growing roots  from the point of mitotic spindle formation until the end of cell division (disappearance of late phragmoplast) was recorded. Results Figure  corrobor Figures . Importa Figures  during t Figures  and firs Figures . Compari Figures , mitotic Figures  and phra Figures . Consist Figures , also du Figures . Based oTime-lapse LSFM imaging of growing roots of transgenic alfalfa plants in 5 min intervals over a period of several hours allowed recording of cells in the meristematic zone and at different stages of cell division. In particular, cells with established PPB (at late G2 phase) were monitored and their entrance to the cell division was documented in time. Preprophase and prophase stage of the cell division was identified from the stage of progressive PPB narrowing when PPB width was in the range of 2–4 μm. This time point has been set up as time 0 min. Recording of preprophase and prophase stage finished by PPB disappearance in cells entering mitosis with starting of mitotic spindle formation in prometaphase. Time-lapse recording of this stage in 5 min intervals revealed its duration for 35–40 min. Interestingly, in roots with different root growth rate this duration was different Figures . In averStage of mitotic spindle was measured from disappearance of the PPB with simultaneous mitotic spindle formation through metaphase and anaphase to mitotic spindle disappearance at telophase. Measurement revealed that termination of the mitotic spindle stage at telophase was different among three groups of roots differing in their growth rate Figures . The aveDuration of the phragmoplast stage was determined from mitotic spindle disappearance at telophase with the formation of early (disk) phragmoplast to disappearance of late (ring and discontinuous) phragmoplast at the end of cytokinesis. Consistent with the duration of previous cell division stages the termination of phragmoplast expansion also occurred at different time among the three groups of roots differing in their growth rate Figures . The aveTo reveal possible relationship between speed of root growth and duration of cell division in root apical meristem we analyzed correlations between duration of individual cell division stages and root growth rates. Three groups of roots with different root growth rates were analyzed separately. Cross-comparison of average root growth rate with average duration of PPB Figure , mitoticCell division is key regulatory component of plant morphogenesis and development and one of the most frequently studied biological processes in the plant biology. Monitoring cell divisions in living multicellular plant organs require real time microscopy approach for long-term observation of intact plants that are adapted to proper physiological conditions. Live cell LSFM imaging of dividing cells could ideally resolve details of cytoskeletal division machinery with high temporal resolution. Moreover, four dimensional monitoring of cell division in organ-, tissue-, and cell type-specific context is required for fundamental understanding of cell division regulation during plant growth and development. Nowadays, different microscopy methods are used to study cell division in genetically, developmentally and microscopically-tractable plant model species. Critically important, however, is the adaptation of currently available microscopy techniques for routine live cell developmental imaging of living robust plant samples, such as crop species. Together with application of efficient protocols for stable transformation of crop species, it should be based on implementation of new approaches, tools and concepts enabling scientific progress in plant cell imaging.ktn1-2 revealed that KATANIN 1 contributes to PPB formation and maturation, and is involved in the positioning of the mitotic spindle and the phragmoplast , End-binding 1 (EB1) proteins, and microtubule severing proteins. Study of MAPs from the MAP65 protein family , an important component of signal transduction cascades in alfalfa (Bekešová et al., Moreover, the present study is the first one to provide large-scale imaging and quantitative characterization of cell divisions in the growing robust root of M. sativa roots favor light-sheet microscopy as the most promising imaging method for the future developmental bioimaging and characterization of robust crop species.Conclusively, previous achievements on Arabidopsis together with data presented in this study demonstrate a high potential of LSFM in developmental live cell imaging of mitotic cytoskeletal arrays in diverse plant species. High temporal and good spatial resolution combined with non-invasive and gentle live-cell imaging of cell divisions during growth and development of MO and PV conducted all live cell imaging. PV made all post acquisition analyses and quantitative evaluations with input from MO and JŠ. MO wrote first draft of the manuscript with input from all co-authors. JŠ conceived and supervised study, helped to interpret data, and finalized manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ligand competition directs heterogeneous bio-chemistry surface and self-assembly for upconversion nanoparticles. via ligand competition reaction. Different binding strengths of phosphodiester bonds and phosphate groups on DNA and the surfactant molecules allow one to create heterogeneous bio-chemistry surface for upconversion nanoparticles. The tailored surface properties lead to the formation of distinct self-assembly structures. Our findings provide insight into the interactions between biomolecules and nanoparticles, unveiling the potential of using nanoparticles as fundamental building blocks for creating self-assembled nano-architectures.Despite significant advances toward accurate tuning of the size and shape of colloidal nanoparticles, the precise control of the surface chemistry thereof remains a grand challenge. It is desirable to conjugate functional bio-molecules onto the selected facets of nanoparticles owing to the versatile capabilities rendered by the molecules. We report here facet-selective conjugation of DNA molecules onto upconversion nanoparticles  In 2005, Costa and co-workers reported that the backbone of DNA molecules can bind to the lanthanide ions,–) on (100)/(010) facets, resulting in selective binding to the two ends of UCNPs; whereas the phosphate group on the end of DNA shows the strongest affinity to replace all the surfactant molecules on UCNPs which creates hydrophilic surface. The location of DNA molecules is experimentally confirmed by analytical chemistry methods and directly visualized by the stochastic optical reconstruction microscopy (STORM). The facet-selective functionalization of UCNPs not only provides insights into the understanding of bio-/nano-interface reaction but also has potential application in self-assembly of structures of nanoparticle building blocks.In this paper, we utilize DNA to investigate the facet-selective binding to the surface of UCNPs. We find that the binding affinity of phosphodiester bonds on the backbone of DNA is stronger than oleic acid (OAH) on (001) facets but weaker than oleate anions  and oleate anions (OA–) on the (001) and (100)/(010) facets of the particles, respectively.To amplify the anisotropic surface properties of UCNPs, instead of using small nanoparticles with small aspect ratios, we choose nanorods of ∼170 nm in length and ∼35 nm in diameter for the study. The ligand exchange reaction is taken place by mixing the rods suspended in chloroform and DNA aqueous solution followed by gentle shaking for 3 hours. e.g. oleic acid, oleate anion, phosphate group and phosphodiester bond, onto the two kinds of facets of the hexagonal prism-like UCNPs based on density functional theory (DFT) simulation (– on (100)/(010) facets thus only replace the OAH molecules on the (001) facets.We compute the binding energies of four different chelating moieties, mulation , table. We propose three scenarios of how the different ligands compete to attach to the anisotropic surfaces of UCNPs , which a+) or hydroxyl (OH–) in the amounts of DNA conjugated to the particles. After ligand exchange, the amount of DNA is quantified by checking the absorbance intensity at 260 nm  in water and preparing them onto the copper grid, only after 5 minutes, two self-assembly patterns, side-by-side  and a 20 minute ultrasonication was applied before dropping 10 μL into a LabTek 8-well chamber immediately for air-drying. The super-resolution images of ATTO-550 conjugated DNA-oligo labelled UCNPs were acquired at 40 Hz for up to 20 000 frames under the excitation of 561 nm laser (10 kW cm–2 at the sample) and activation of 405 nm laser (≤5 kW cm–2 at the sample). The excitation beams were reflected by a custom-designed polychroic mirror . Fluorescence emissions from ATTO-550 were filtered by a bandpass filter . An imaging buffer  with an oxygen scavenger system  was used for the STORM imaging. STORM images were analyzed using Insight3  for single-molecule localization and custom-written Matlab codes for cluster analysis based on nearest-neighbour algorithm.The Stochastic Optical Reconstruction Microscopy (STORM) imaging of UCNPs was carried out with Olympus cellTIRF-4Line system . After conjugated with ATTO-550 labelled DNA molecules, the UCNPs water suspension was diluted for 1000 times (2.5 × 10There are no conflicts to declare.Supplementary informationClick here for additional data file.


In quantitative biology, mathematical models are used to describe and analyze biological processes. The parameters of these models are usually unknown and need to be estimated from experimental data using statistical methods. In particular, Markov chain Monte Carlo (MCMC) methods have become increasingly popular as they allow for a rigorous analysis of parameter and prediction uncertainties without the need for assuming parameter identifiability or removing non-identifiable parameters. A broad spectrum of MCMC algorithms have been proposed, including single- and multi-chain approaches. However, selecting and tuning sampling algorithms suited for a given problem remains challenging and a comprehensive comparison of different methods is so far not available.We present the results of a thorough benchmarking of state-of-the-art single- and multi-chain sampling methods, including Adaptive Metropolis, Delayed Rejection Adaptive Metropolis, Metropolis adjusted Langevin algorithm, Parallel Tempering and Parallel Hierarchical Sampling. Different initialization and adaptation schemes are considered. To ensure a comprehensive and fair comparison, we consider problems with a range of features such as bifurcations, periodical orbits, multistability of steady-state solutions and chaotic regimes. These problem properties give rise to various posterior distributions including uni- and multi-modal distributions and non-normally distributed mode tails. For an objective comparison, we developed a pipeline for the semi-automatic comparison of sampling results.The comparison of MCMC algorithms, initialization and adaptation schemes revealed that overall multi-chain algorithms perform better than single-chain algorithms. In some cases this performance can be further increased by using a preceding multi-start local optimization scheme. These results can inform the selection of sampling methods and the benchmark collection can serve for the evaluation of new algorithms. Furthermore, our results confirm the need to address exploration quality of MCMC chains before applying the commonly used quality measure of effective sample size to prevent false analysis conclusions.The online version of this article (doi:10.1186/s12918-017-0433-1) contains supplementary material, which is available to authorized users. In the field of computational systems biology, mechanistic models are developed to explain experimental data, to gain a quantitative understanding of processes and to predict the process dynamics under new experimental conditions –3. The pThe parameters of biological processes are often estimated using frequentist and Bayesian approaches , 5. FreqFor optimization, a large collections of benchmark problems were established to facilitate a fair comparison of methods . FurtheAdaptive - Metropolis , state vector f and the initial conditions x0(η) define the temporal evolution of the state variables as functions of η. For biological processes, experimental limitations usually prevent the direct measurement of the state vector x(t). Instead, measurements provide information about the observable vector y(t). The observables depend on the state of the process, y=h, in which h denotes the output map f can be found in “with time y yields noise corrupted experimental data The measurement of the observables σ denotes the standard deviation of the measurement noise and with i=1,…,ny. An example for noisy measurement data is discussed and visualized in the “in which σ are usually unknown and part of the parameter vector, i.e., θ=. The likelihood of observing the data θ is The standard deviations y(tk) depends implicitly on η.in which In Bayesian parameter estimation the posterior p(θ) denotes the prior and is considered, in which θ given the experimental data p(θ) ))36]) as To facilitate a comprehensive evaluation of sampling methods, we considered the aforementioned benchmark problems (M1-6). These benchmark problems possess a wide range of different properties regarding the underlying dynamical system (e.g. mono- and bistable) as well as the posterior distribution . This renders the collection presented suitable for the in-depth evaluation and will facilitate the derivation of guidelines for the a priori selection of the appropriate sampling scheme.6 iterations. The large amount of sampling results was analyzed using the analysis pipeline illustrated in Fig. We sampled the posterior distributions of all benchmark problems using the algorithms introduced in the “Given the sampling results, we asked the question how EQ depends on the benchmark problem and its properties. We found that the size of the groups of runs identified by the analysis pipeline Fig. a and theIn terms of the dynamical properties of the underlying dynamical system, our results for the benchmark problems indicated that state-of-the-art sampling methods work well with multiple steady states and saddle-node bifurcations, as well as Hopf bifurcations and (limit cycle) oscillations resulting in weak multi-modality of the posterior, oscillating trajectories. However, these methods still fail in case of (aperiodic) oscillations/chaotic behavior and local non-identifiability resulting in strong multi-modality of the posterior.The analysis on the level of sampling methods revealed that for (M2-4) most algorithms worked appropriately Fig. b while fFollowing the analysis of the differences between benchmark problems, we compared single- and multi-chain methods. The average performance characteristics for single- and multi-chain methods were computed by averaging over sampling methods, initialization schemes and tuning parameter choices Fig. . We founIn addition to characteristics of methods, we assessed the importance of initialization schemes. Therefore, the average performance characteristics for RND and MS initialization were computed by averaging over sampling methods and tuning parameter choices Fig. . This reA detailed analysis revealed that some methods were more sensitive to the initialization than others. The performance of PT appeared to be almost independent of the initialization scheme Fig. b, makingTo provide guidelines regarding tuning parameters and adaptation mechanisms, we carried out a fine-grained analysis of sampling method and subclasses of them. The assessment of single-chain samplers revealed that the adaptive Metropolis methods with acceptance rate dependent proposal scaling (AM(acc)) outperformed methods with dimension-dependent proposal scaling (AM(dim) and DRAM(dim)) as shown in Fig. The PT algorithms employed in this study used temperature and proposal density adaptation. We evaluated different swapping strategies and strategies to select the number of temperatures. The best performance characteristics were achieved with a large, fixed number of temperatures  based algorithms such as NUTS or Wormhole Monte Carlo , 78, regIn summary, our comprehensive evaluation revealed that even state-of-the-art MCMC algorithms have problems to sample efficiently from many posterior distributions arising in systems biology. Problems arose in particular in the presence of non-identifiabilities and chaotic regimes. The examples provided in manuscripts presenting new algorithms are often not representative and a more thorough assessment on benchmark collections should be required (as is common practice in other fields). The presented study provides a basis for future developments of such benchmark collections allowing for a rigorous assessment of novel sampling algorithms. In this study, we already used six benchmark problems with common challenges to provide practical guidelines for the selection of sampling algorithms, adaptation and initialization schemes. Furthermore, the presented results highlight the need to address chain exploration quality by taking into account multiple MCMC runs which can be compared with each other before calculating effective sample sizes. The availability of the code will simplify the extension of the methods and the extension of the benchmark collection.Additional file 1Supplementary Notes. Covering additional details about the analysis pipeline and sampling results. (PDF 1200 kb)Additional file 2Supplementary Code. Containing a standalone implementation of methods, benchmark problems, data sets and analysis tools used in this study. (ZIP 6953 kb)


HLA antibodies are primary causes of transplant rejection; they recognize epitopes that can be structurally defined by eplets. There are many reviews about HLA epitope-based matching in transplantation. This article describes some personal reflections about epitopes including a historical perspective of HLA typing at the antigen and allele levels, the repertoires of antibody-verified HLA epitopes, the use of HLAMatchmaker in determining the specificities of antibodies tested in different assays, and, finally, possible strategies to control HLA antibody responses. EacAlthough CREG matching has been applied to the identification of compatible platelet donors for refractory alloimmunized thrombocytopenic patients , no beneSerological HLA typing had always accuracy problems because of the lack of reagents and technical limitations. After elucidation of the HLA molecular structure and nucleic acid sequencing of HLA antigens during the late 1980s, the application of DNA-based technologies permitted accurate HLA antigen typing results. Very soon, many antigens were identified with amino acid differences, and this led to assignments of alleles to be annotated with a colon (:) followed by two and later on three digits after more than 100 alleles corresponding to the 2-digit antigen had been identified (Examples are A*02:01 and A*02:101). Certain amino acid differences affect the expression of eplets. For instance, A*24:02 has the antibody-verified 166DG eplet shared with A*01:01, A*23:01, A*80:01, and B*15:12, whereas A*24:03 has 166EW shared with A*02:01, A*03:01, etc. This example illustrates the difficulty of matching at the antigen level, in this case A24 if the patient has antibodies against 166DG or 166EW. It is now recognized that HLA compatibility is better determined at the allele than at the antigen level .There are thousands of HLA alleles and the question arises which ones should be typed for in the clinical transplant setting. One might focus on alleles present in the patient and donor population of a given transplant program. Although rare alleles might be excluded it is now apparent that because of the increasing racial and ethnic heterogeneity of most populations such alleles occur more frequently as mismatches. Tissue typing techniques are moving forward very fast in the clinical setting, and each HLA allele has precise information about its eplet repertoire. The degree of eplet mismatching of a donor allele depends on the HLA phenotype of the recipient, which has its own repertoire of self-eplets. Such determinations can be readily made with specifically designed computer programs such as HLAMatchmaker.Epitope-based HLA compatibility determination requires a basic understanding of how antibodies interact with epitopes. Antibody reactivity testing with HLA panels can be done with different techniques from immunoglobulin and complement component C1q binding to isolated HLA molecules attached to microbeads to flow cytometric binding on lymphocytes and complement-dependent lymphocytotoxicity. These methods may give different results which make clinical relevance assessments difficult. Epitope specificity analyses of antibodies may clarify some of these issues.Antibody binding to protein epitopes occurs through six complementarity-determining region (CDR) loops, three of them H1, H2, and H3 are on the immunoglobulin heavy chain and L1, L2, and L3 are on the light chain. Each loop interacts with a small set of amino acid residues in the so-called structural epitope and CDR-H3 which binds to the so-called functional epitope (or hot spot) in a central location has a dominant role in determining antibody specificity. Eplets are considered to be equivalent to functional epitopes. Figure Antibodies through their CDR-H3 recognize specific eplets centrally located in the structural epitope. Other eplets in the same sequence location but with different residue compositions are generally non-reactive, but there can be exceptions if the eplet has a high degree of residue homology with the eplet that had induced the antibody. As an example, the 82LR eplet, which describes the well-known Bw4 epitope, has two closely nearby residues 80I and 80T. Many 80I + 82LR-induced antibodies recognize also 80T + 82LR and binding strengths can vary from high to low. This is an example of the so-called Landsteiner type of serological cross-reactivity, whereby different but structurally related epitopes react with the same antibody. It should be noted that certain 80I + 82LR-induced antibodies never react with 80T + 82LR; they can be designated as specific for only 80I. This epitope-based analysis seems helpful in the assessment of the mismatch acceptability of 82LR (or Bw4) when 80T + 82LR-carrying alleles have no or very low reactivity with patient’s serum.As illustrated in Figure As illustrated in Figure For many protein antigen–antibody complexes, there is a certain level of permissiveness for residue substitutions in critical contact areas, and this applies also for HLA epitopes. Certain residue substitutions in the structural epitope have a moderate effect on an allele’s reactivity with antibody, whereas others are inhibitory to the level of weak reactivity or non-reactivity of specific eplet-carrying alleles. The latter might be considered as epitope-based acceptable mismatches.The same rules apply to epitope descriptions of class II alleles which more often solely correspond to eplets. Only few DRB epitopes correspond to eplet pairs, and this might be due to monomorphic residue nature of the DRA chain. Several immunogenic DRB1 eplets are also on alleles encoded by DRB3, DRB4, and/or DRB5. Each of them has distinct antibody-verified eplets, and it seems that epitope matching for DRB3/4/5 affects the class II antibody response.DQ and DP encode for heterodimers of A and B chains which are both polymorphic and have distinct eplets many of which have been antibody verified. DQ-specific antibodies appear most prevalent among antibodies induced by class II mismatches. Such antibodies are specific for eplets on DQA and DQB chains, and there is emerging evidence that some DQ epitopes are defined by eplet pairs involving both chains , 19. Thiwww.epitopes.net (formerly www.HLAMatchmaker.net) has now a downloadable Excel document “Five Maps of HLA Epitopia,” which describes the sequence locations of antibody-verified eplets and polymorphic residues as potential candidates defining additional epitopes. These maps can be used in navigating the continents of HLA Epitopia while searching for newly antibody-defined epitopes  epitopes, which make detections of antibodies against lower frequency epitopes more difficult unless these antibodies are separated through absorption–elution studies with selected alleles.Technique dependencies of serum reactivity may also affect the interpretation of epitope specificities, especially for highly sensitized patients who have several antibody populations in different concentrations and affinities that affect their reactivity with HLA panels. Again, absorption–elution studies with selected alleles might dissect these serum reactivity patterns, so that an epitope analysis can be more readily done.The HLAMatchmaker website has three downloadable antibody analysis programs in Excel format: HLA-ABC, HLA-DRDQDP, and MICA. The latest 02 versions focus on antibody-verified epitopes recorded so far in the HLA Epitope Registry. All of them correspond to single eplets or eplets paired with other residue configurations uniquely shared by a group of antibody-reactive alleles. The antibody analysis programs also include “other” theoretical eplets, which might become experimentally verified if informative antibodies are identified. The HLAMatchmaker website has a downloadable instruction manual for the epitope analysis of HLA antibodies tested in assays with single alleles.The 02 versions require entering of the HLA information of the panel, the MFI values of each allele and the allelic HLA type of the patient and preferably the immunizer . A calculation of the MFI for the self alleles offers a basis for determining a cutoff value be entered, and the program then determines which epitopes are shared between reactive alleles.In the clinical setting, the primary purpose of the serum HLA antibody analysis of transplant candidates is to identify potential donors whose mismatched HLA alleles are acceptable. This approach is useful not only for organ transplantation but also for platelet transfusions of allosensitized thrombocytopenic patients. Eurotransplant has incorporated HLAMatchmaker in the Acceptable Mismatch Program to identify donors for highly sensitized patients , 24.Epitope specificity analyses might also be useful in desensitization protocols to remove donor-specific antibodies . Such prMany posttransplant studies have shown associations between the appearance of donor-specific antibodies with allograft rejection and failure. Most studies have cases whereby the transplant continues to function quite well in the presence of donor-specific antibodies, and this raises the question which antibodies are clinically significant in transplantation. By definition, such antibodies must be absorbed by the allograft where they recognize epitopes expressed on the vascular endothelium and other tissues and initiate inflammatory processes leading to rejection. Accordingly, testing for circulating HLA antibodies in the presence of a transplant has its limitations, and this becomes apparent with the increased serum reactivity often seen after allograft nephrectomy –28, and HLA matching at the epitope level also benefits transplant outcome in non-sensitized patients who have no donor-specific HLA antibodies before transplantation. Numerous studies have demonstrated significant correlations between eplet loads of HLA mismatches and the development of donor-specific class I and class II antibodies as well as rejection incidence and allograft outcome –48. ThesSecond, eplet loads can be used to develop new donor selection strategies for non-sensitized recipients especially younger patients. Some transplant programs have already begun to implement this approach –51. HLA The relative immunogenicity of HLA eplets plays an important role in mismatch permissibility of HLA alleles. This can be determined empirically by analyzing the frequencies of antibody responses to mismatched donor eplets which depend on the HLA phenotypes of the recipient and their specificities would be for epitopes defined by eplets or eplet pairs . Such stThree recent concepts have begun to address these mechanisms. First, the non-self–self paradigm of HLA epitope immunogenicity is based on the hypothesis that HLA antibodies originate from B-cells with low-avidity, self-HLA epitope-specific Ig-type receptors that can interact with non-self eplets to generate a signal for B-cell activation. Such non-self eplets would be recognized by the specificity-determining CDR-H3, and in the context of the corresponding structural epitope they must be surrounded by self-residues that contact the other CDRs of antibody –56. SecoThese are reasonable theoretical concepts, although no direct experimental methods are available to analyze the very early phases of the antibody response . At presEpitope-based HLA matching has become a new concept in the clinical transplant setting. It relies on HLA typing at the allele level and can be used to identify acceptable mismatches for sensitized patients and to develop permissible mismatch strategies for non-sensitized patients. Our understanding of HLA epitopes is still in progress, and more studies are needed to identify antibody-verified epitopes to be recorded in the HLA Epitope Registry. Also, histocompatibility testing laboratories will have opportunities to sort out complex serum reactivity patterns and interpret technique-dependent epitope specificities of HLA antibodies and their clinical relevance. Sooner or later, there will be new epitope-based HLA-matching approaches with increased benefits to transplant patients.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Early development of neurocognitive functions in infants can be compromised by poverty, malnutrition and lack of adequate stimulation. Optimal management of neurodevelopmental problems in infants requires assessment tools that can be used early in life, and are objective and applicable across economic, cultural and educational settings.The present study examined the feasibility of infrared eye tracking as a novel and highly automated technique for assessing visual-orienting and sequence-learning abilities as well as attention to facial expressions in young (9-month-old) infants. Techniques piloted in a high-resource laboratory setting in Finland (N=39) were subsequently field-tested in a community health centre in rural Malawi (N=40).Parents' perception of the acceptability of the method  and percentages of infants completing the whole eye-tracking test  were high, and percentages of valid test trials  satisfactory at both sites. Test completion rates were slightly higher for eye tracking (90%) than traditional observational tests (87%) in Malawi. The predicted response pattern indicative of specific cognitive function was replicated in Malawi, but Malawian infants exhibited lower response rates and slower processing speed across tasks.High test completion rates and the replication of the predicted test patterns in a novel environment in Malawi support the feasibility of eye tracking as a technique for assessing infant development in low-resource setting. Further research is needed to the test–retest stability and predictive validity of the eye-tracking scores in low-income settings. Optimal management of neurodevelopment problems in infants requires assessment tools that are applicable across cultural, educational, and economic settings.Eye-tracking applications have been developed for the assessment of visual and cognitive functions in high-resource laboratories.This study demonstrates the feasibility of eye tracking in rural Malawi, supporting its use as a technique for assessing infant cognitive development in low-resource settings.Human brain development is highly dependent on access to ‘optimal’ environment during the first years of lifeExisting techniques for assessing early cognitive development in infantsAutomated eye-tracking-based tests have been developed for assessing infants' visual acuity,17Eye-tracking technologies have been extensively used in Europe, North America and Japan, but there is a paucity of studies using this method in low-income countries. Cultural differences, such as unfamiliarity with the concept of infant cognitive testing, lack of experience of screen-based stimuli and test settings, could interfere with the method being successfully implemented in low-income settings.We examined the feasibility of eye tracking as a novel technique for assessing infants' cognitive function in low-resource settings. We created a battery of tests that have been previously used in high-resource settings, piloted the test battery in a high-resource laboratory in Finland and subsequently tested it in a rural low-income environment in Malawi. As a direct test of feasibility, we compared the two test sites for (1) parents' perception of the acceptability of the method, (2) test completion rates and (3) patterns of infants' test responses. Similar comparisons were conducted for traditional observational assessments of infant cognition. For the eye-tracking method to be defined as feasible, we expected (1) that most parents accepted the method in Finland and Malawi, and test completion rates in Finland and Malawi were comparable and within the range reported in previous studies . The study protocol was approved by the ethics committees of the College of Medicine, Malawi, Pirkanmaa Hospital District and University of Tampere, Finland. Written informed consent was obtained from the participants' biological mother.Eye-tracking assessment took place in a quiet and dimly lit room . The infEye-tracking data were analysed using automated MATLAB scripts  and similar criteria as those used in prior studies.Traditional structured observation tests were administered to assess infants' ability to respond to and initiate non-verbal social communication.10.1136/archdischild-2016-310525.supp1Supplementary dataMothers were asked (1) whether they had enjoyed participating with their child in the assessment and (2) whether they would recommend a friend to participate in the assessment with their child. The responses were marked as ‘yes’ or ‘no’.Descriptive data were collected by interviewing the mothers using a socio-demographic questionnaire, including questions about maternal age, parental education level and family structure. Infants' length was measured to the nearest 1 mm by using a length board  and infant weight by using an electronic infant weighing scale (SECA 735) with reading increments of 10 g. Mid upper arm circumference (MUAC) and head circumference were measured to the nearest 1 mm with non-stretchable plastic insertion tapes. Anthropometric measurements were done in triplicate by trained personnel, and the mean of the triplicate measurements was used to calculate age-standardised and sex-standardised anthropometric indices  using the WHO standards.242 tests for dichotomous variables , t-tests for normally distributed continuous variables and non-parametric Mann-Whitney U tests for non-normal continuous variables.The samples were compared by using Pearson's χA total of 39 Finnish and 40 Malawian infants were enrolled for the study in May/June 2014 (Finland) and September 2014 . Three of the Malawian infants did not participate in eye tracking because their mothers opted out (n=2) or because the assessments could not be conducted . No data were obtained from these participants, but parental acceptance rates were calculated by including the two mothers who opted out, and test completion rates by including the infant who was not assessed due to fussiness . All other statistics, including sample descriptives , percentThe Finnish and Malawian infants assessed with eye tracking did not differ in sex ratios or age, but significant group differences were found in socio-demographic and anthropometric variables .In total, 95% (N=37) of the Finnish mothers and 92% (N=36) of the Malawian mothers reported that they enjoyed participating in the eye-tracking assessment with their child, and 100% (N=39) of Finnish and 95% (N=37) of Malawian mothers stated they would recommend a friend to participate with their child. And 95% (N=37) of the Finnish and 89.5% of the Malawian (N=34) infants completed the whole test battery (∼15–25 min of testing). For five infants, the assessment was terminated because the infant became too tired/inattentive , a discontinuation in electricity supply  or the infant reacted negatively to the stimuli in one task . These five infants were still able to provide sufficient number of valid trials to meet the inclusion criteria for most of the eye-tracking tasks.The completion rates for the eye-tracking tasks were slightly higher in Finland than in Malawi, although none of the differences were significant  at both sites and largely comparable to the corresponding values for traditional observational tests. Compared with Finnish infants, Malawian infants had lower percentage of valid test trials in two out of three eye-tracking tasks. The percentage of valid trials were below the expected range  for one task in Finland and two tasks in Malawi. The deviations from the expected range were, however, relatively small in magnitude at both sites (<4%) and unlikely to have practically meaningful consequences for the feasibility of eye tracking. Together, these results suggest that there are no barriers for conducting eye tracking in low-income environments.As an additional indicator of transferability of eye tracking, our results showed similar pattern of responses in infants at the two sites. Similar to Finnish infants, Malawian infants exhibited active visual search, anticipatory saccades and prioritised attention to facial expressions. This result suggests that the cognitive constructs that have been documented in studies in Europe, the USA and JapanCompared with Finnish infants, Malawian infants had a lower rate of successful visual search responses, longer attention shift times (processing speed) and a lower rate of gaze disengagements from faces. While these findings could be interpreted as an indication of a cognitive disadvantageOur study did not have adequate power to detect small to medium differences (Cohen's d<0.8) in feasibility indices between the sites. It is unlikely, however, that more subtle differences not detected in this study would create major challenges for the implementation of the test. The current study was also limited to one low-income setting. Whereas we consider it unlikely that the implementation of eye tracking would be markedly different in other low-income settings, such heterogeneity cannot be ruled out. These limitations notwithstanding, the present study provides a demonstration of the feasibility of eye tracking in low-resource setting and motivates further studies to examine individual variations in eye-tracking test scores, their psychometric properties and, ultimately, predictive value in low-income settings.


The antenatal diagnosis of vascular abnormalities such as coarctation of the aorta may allow for more timely provision of what can be life-saving postnatal care. Fetal MRI offers the potential to compliment conventional antenatal assessment of the extracardiac vasculature, which can be difficult to assess by ultrasound.Using overlapping multi-slice 2D single-shot fast spin echo sequences  in a fetus at 36 weeks gestation, we applied a novel GPU accelerated super-resolution algorithm for slice-volume registration to the oversampled data to compensate for fetal movements between slices.A 3D dataset was generated with an isotropic voxel size of 0.4 mm for visualisation of fetal structures. Using this we were able to clearly show the relationship of the great vessels, demonstrating hypoplasia of the aortic arch and a high risk of coarctation. Following delivery, critical coarctation of the aorta was confirmed. The baby went on to have surgical repair in the neonatal period.Uncontrolled fetal movement represents a major challenge to more widespread adoption of fetal cardiovascular MRI. Automated motion-corrected 3D volume reconstructions could greatly increase the diagnostic utility of antenatal MRI in the future.


Also, response latency and the corticomuscular coherence (CMC) time delay were affected (p < 0.05) by anodal tDCS when compared to sham tDCS. Furthermore, gray box simulation results from a Simplified Spinal-Like Controller (SSLC) model demonstrated that the input-output function for motor evoked potentials (MEP) played an essential role in increasing muscle activation levels and response time improvement post-tDCS when compared to pre-tDCS baseline performance. This computational approach can be used to simulate the behavior of the neuromuscular controller during VMT to elucidate the effects of adjuvant treatment with tDCS.Induction of neuroplasticity by transcranial direct current stimulation (tDCS) applied to the primary motor cortex facilitates motor learning of the upper extremities in healthy humans. The impact of tDCS on lower limb functions has not been studied extensively so far. In this study, we applied a system identification approach to investigate the impact of anodal transcranial direct current stimulation of the leg area of the motor cortex via the human visuo-myoelectric controller. The visuo-myoelectric reaching task (VMT) involves ballistic muscle contraction after a visual cue. We applied a black box approach using a linear ARX (Auto-regressive with eXogenous input) model for a visuomotor myoelectric reaching task. We found that a 20th order finite impulse response (FIR) model captured the TARGET (single input)—CURSOR (single output) dynamics during a VMT. The 20th order FIR model was investigated based on gain/phase margin analysis, which showed a significant ( Non-invasive brain stimulation (NIBS) techniques, such as transcranial direct current stimulation (tDCS)—an electrically based intervention—can modulate brain activity to promote neuroplasticity , the EMG of the TA muscle, as shown in Figure We first leveraged a linear black-box model, the linear Auto-regressive with eXogenous input (ARX) model, which is the simplest input and output polynomial model that can be fitted using the System Identification toolbox of Matlab . In the ARX model, the parameters of the error term are linear, and the linear least squares method can be used for parameter estimation. This method is numerically simple and reliable and avoids problems of local minimum and convergence. This ARX model can be used to investigate the transfer function using its gain/phase margin. Then, for gray-box modeling  and female (6) volunteers (age: 26 ± 5 years) participated in this study after giving informed consent, and all experiments were conducted in accordance with the Declaration of Helsinki. The study obtained ethics approval at the University Medical Center, Goettingen, Germany. Subjects who (i) were taking any acute or regular CNS-active medication, and (ii) had a history of neurological, psychiatric, or medical disease, family history of epilepsy, pregnancy, cardiac pacemaker or previous surgery involving metallic implants at the time of the study, were excluded from participation.http://psychtoolbox.org/) GUI—see bottom panel of Figure http://www.ced.co.uk/pru.shtml?sig3wglu.htm) via a 16-bit DAQ for offline analysis. Cortico-spinal excitability was monitored before and after tDCS with TMS  for VMT, as shown in Figure Our goal was to perform a system identification using the FIR model and its gain/phase margin analysis to find out how performance of the human myoelectric controller changed during visuomotor task performance due to tDCS (comparing Pre-tDCS and Post-tDCS VMT sessions). We first investigated the human visuo-myoelectric controller using a black box system identification approach, specifically a single-input single-output polynomial model (“polyest” in Matlab). Trial-to-trial variation was investigated to understand how the brain optimized response time for the initially ballistic (open-loop) task component, and response accuracy moderated by visual feedback during the myoelectric control task component. We fitted a FIR model between the TARGET and the CURSOR. A respective block diagram for VMT is shown in Figure eT(s) is defined by:D is the time delay from the cortical centers to the spinal centers, f (t– δ), and whose Laplace Transform is represented by,es−δ. Thus, for the tibialis anterior muscle,We simulated the human visuo-myoelectric controller  function of the primary pyramidal neurons  VMT data in a closed-loop condition, the SSLC will need visual feedback   and the real value . Based on the direction of the calculated error of the two signals, the gains change iteratively until the error no longer changes, or is within a user-defined tolerance—typically within 5%. Therefore, the SSLC model fits to subject-specific pre-tDCS TMS-MEP SR curve and VMT data, which will be able to predict post-tDCS VMT data based on the post-tDCS TMS-MEP SR curve.post-hoc tests, Student's t-tests were applied.EMG response latency was determined offline as the period from the instant of the visual TARGET cue to the moment when the rectified EMG in a sliding window of 500 ms from the TA muscle was more than three times of the standard deviation of the resting value . The CURSOR response latency was determined offline as the duration from the instant the visual TARGET cue was provided for ballistic muscle contraction to the time the CURSOR crossed the TARGET during VMT. CURSOR response accuracy was computed as Root Mean Square Error (RMSE) between the CURSOR and the TARGET signals during the 5 s of TARGET cue presentation. We investigated post tDCS differences in EMG response latency, RMSE, CURSOR response latency, CMC peak, and CMC time delay from pre-tDCS baseline values using a two-way  ANOVA. In addition, post-tDCS changes in gain/phase margins from pre-tDCS baseline values were identified by a two-way  ANOVA. For p < 0.05), however not on the phase margin (p = 0.143). The trial effects and the interaction between trial and group were not significant for gain margin  as well as phase margin . Also, gain margin  however not significant for phase margin (p = 0.143). In addition, there was a significant main effect of group (tDCS/sham) on the EMG response latency, CURSOR response latency as well as CMC time delay (p < 0.05). However, the main effect of the factor group (tDCS/sham) on the RMSE and the beta-range CMC peak was not significant.ARX models using least squares with regularization were estimated with the help of the system identification toolbox in Matlab . The 20th order FIR model was found to be suitable across subjects based on the AIC criterion. Figure Figure p < 0.05) increase in gain margin was found when compared to sham tDCS , we found that anodal tDCS induced a statistically significant CMC enhancement of the tibialis anterior muscle during quiet standing with eyes closed, 45 and 60 min after the end of tDCS. Therefore, the effect of anodal tDCS on CMC needs further investigation with larger sample sizes.Based on the computational modeling, we hypothesized that anodal tDCS enhances the responsiveness of ePN to afferent feedback  and improved response time (correlate of CURSOR response latency) of task performance. This suggests that the muscle response frequency , as well as the gain of the urgency of the respective movement increase . After severe demyelination, conduction speeds can drastically reduce leading to significant signal delay times, where es−δ approaches zero so that only the co-contraction remains:Regarding clinical relevance of the computational results, the change in the slope of the I-O curve of ePN may be able to compensate for small changes in the signal conduction speeds  that needs further investigation. We specifically modeled co-contraction in the SSLC since Roche and colleagues showed that anodal tDCS induces effects on spinal network excitability similar to those observed during co-contraction, suggesting that anodal tDCS activates descending corticospinal projections mainly involved in co-contractions (Roche et al., VK carried out the computational modeling under the guidance of FS and AD. AF, SB, and MN substantially contributed to the neurophysiological interpretation of the computational results. FS, AD, AF, SB, and MN have substantially contributed to the scientific analysis of this work. All authors have also drafted the work and revised it critically with contribution related to author order. All authors have approved the final version before submission.MN is member of the Scientific Advisory Board of Neuroelectrics. The other 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.


Adaptive immunity is the specialized defence mechanism in vertebrates that evolved to eliminate pathogens. Specialized lymphocytes recognize specific protein epitopes through antigen receptors to mount potent immune responses, many of which are initiated by nuclear factor-kappa B activation and gene transcription. Most, if not all, pathways in adaptive immunity are further regulated by post-translational modification (PTM) of signalling proteins, e.g. phosphorylation, citrullination, ubiquitination and proteolytic processing. The importance of PTMs is reflected by genetic or acquired defects in these pathways that lead to a dysfunctional immune response. Here we discuss the state of the art in targeted proteomics and systems biology approaches to dissect the PTM landscape specifically regarding ubiquitination and proteolysis in B- and T-cell activation. Recent advances have occurred in methods for specific enrichment and targeted quantitation. Together with improved instrument sensitivity, these advances enable the accurate analysis of often rare PTM events that are opaque to conventional proteomics approaches, now rendering in-depth analysis and pathway dissection possible. We discuss published approaches, including as a case study the profiling of the N-terminome of lymphocytes of a rare patient with a genetic defect in the paracaspase protease MALT1, a key regulator protease in antigen-driven signalling, which was manifested by elevated linear ubiquitination.This article is part of the themed issue ‘Quantitative mass spectrometry’. As a first line of defence, the relatively unspecific innate immune system detects structural components of microorganisms termed pathogen-associated molecular patterns (PAMPs). Innate immune cells are equipped with a range of pattern recognition receptors (PRRs) such as the Toll-like receptor family that activates immune signalling cascades within the cells upon ligation of the PAMP receptor, leading to an inflammatory response and elimination of the invading pathogen (reviewed in ). The se2.Ubiquitination is a common and reversible PTM where the 8.5 kDa ubiquitin protein is ligated to a target lysine residue in a substrate protein. This process is orchestrated by a coordinated three-step reaction of ubiquitin-activating (E1), ubiquitin-conjugating E2) and ubiquitin-ligating E3) enzymes [ and ubiq enzymes Regulated ubiquitination of key substrates is critical in most signalling pathways in both innate and adaptive immunity (reviewed in ), includ3.Proteomic profiling of interactors with known signalling proteins has identified unexpected ubiquitin-related proteins, painting a picture of a far more complicated and intertwined regulatory mechanism than often assumed. Interactors of E3 ligase Cbl and its known interactor CIN85 associate with the phosphatase SHIP-1 upon BCR cross-linking, epitomizing cross-talk between phosphorylation and ubiquitination . Mass spThe crucial role of ubiquitination in immunity, combined with knowledge gaps, makes this system an interesting target for unbiased quantitative proteomics profiling of antigen receptor signalling. Ubiquitin proteomics has been hard to achieve. The high proteolytic activity of DUBs makes preserving the ubiquitination status after cell disruption challenging, and due to the low relative abundance of specific ubiquitination events compared with the total protein pool, a selective enrichment of ubiquitinated proteins is desired prior to MS analysis. Ubiquitin-modified proteins yield specific peptides upon tryptic digestion of the proteome; the modified lysine residue will retain a diglycyl isopeptide remnant that can be identified by MS. Two potential pitfalls are present, however; first, the diglycyl remnant is not unique to ubiquitinated proteins but also results from proteins modified with the less abundant ubiquitin-like modifiers interferon-stimulated gene-15 (ISG15) and NEDD8, so further validation is required. Secondly, the mass shift due to the ubiquitin remnant is identical to an artefact modification of lysine occurring during carbamidomethylation to block thiol groups on cysteine residues, which therefore requires careful sample handling, temperature control and data analysis .a and reviewed in [Affinity enrichments of the pool of ubiquitinated proteome (ubiquitome) followed by MS analysis have been applied with varying degrees of success (figure 2iewed in ). Combiniewed in , but as iewed in . One couiewed in . Combiniiewed in ) or receiewed in ) can proSeveral approaches have used DUBs to probe the ubiquitinated proteome. By using a set of DUBs with assumed chain topology specificity, ubiquitin chain restriction (UbiCRest) provides the opportunity to investigate the different homo- and heterotypic polyubiquitin chains that are conjugated to substrates . The GevThe largest breakthrough in ubiquitin proteomics was achieved by the generation of a specific antibody against the diglycyl-lysine group that results from tryptic digestion of a ubiquitinated protein to allow enrichment of remnant containing peptides –28. By uThis was an interesting finding, since linear ubiquitination has been reported in many other immune signalling pathways, but was never assumed to be relevant in antigen receptor stimulation. Linear ubiquitination of substrates like NEMO seems to play an important role in scaffolding of the receptor signalosome, and the identification of BCL10 as an additional target in this pathway opens up the possibility of a much larger role for LUBAC in lymphocytes than previously thought. Many of the E3 ligases (such as the TRAF family) share a common role in different pathways, even though stimulation may result in different cellular outcomes. Analysis of potential differential substrate specificity depending on the receptor that is triggered could provide interesting biological insights.4.Proteolysis is an irreversible PTM  and is a+ cytotoxic T cells, and caspases regulate both maturation of lymphocytes and regulated removal of the expanded specific lymphocyte population by apoptosis after the immune challenge has resolved  presentation of antigens by antigen-presenting cells (APCs) depends on regulated proteolysis of intracellular or internalized proteins into MHC-presentable peptides by the immuno-proteasome (for MHC-I) or proteolysis by legumain and cathepsins (in case of MHC-II). Legumain also activates the MHC-II protein complex inside the MHC compartment, to allow loading of the newly generated peptide (reviewed in ). Proteaiewed in ). MALT1 iewed in ). Finalliewed in .in vivo; proteases can be translated as inactive zymogens that require removal of a pro-peptide for activation, and there is an extensive repertoire of endogenous inhibitors that restrict proteolysis. This makes the study of proteases by proteomics techniques challenging [Protease function is highly regulated llenging . Mere qullenging .5.Since the function of a protease is defined not only by its activity status but particularly by the proteins it cleaves, several groups have dedicated their efforts to developing proteomics techniques for unbiased identification of protease substrates . As b). Positive selection of the neo-N-terminal peptide after chemical labelling of the N-α-amino group by NHS-biotin seems like an attractive technique, but since normal chemical labelling conditions for α-amino groups will inadvertently also lead to modification of the lysine ϵ-amino group, specialized techniques are required to introduce selectivity. The Wells lab developed an elegant technique for selectively labelling α-amines with biotin by using a subtiligase enzyme to transfer a biotinylated peptide sequence containing a tobacco etch virus (TEV)-protease cleavage site selectively to the unmodified N-terminus of proteins [Since bona fide substrates are uniquely identifiable by the novel or neo-N-terminal protein end of the cleavage fragment, methods that select for and enrich this peptide have been most successful  and in vitro by granzyme-B, or cells exposed to natural killer cells, were analysed by COFRADIC [COFRADIC has been applied in several studies using lymphocytes to investigate substrates of members of the granzyme protease family. Granzymes are effector proteases used by cytotoxic T cells to neutralize pathogen-infected cells; upon recognition of MHC-presented antigen on an APC, cytotoxic T cells secrete granules containing granzymes and perforin that enter the APC and induce cell death. Lysates of SILAC labelled Jurkat T cells pretreated with pan-caspase inhibitors and then digested COFRADIC . More thCOFRADIC . DiffereCOFRADIC ,65. COFRCOFRADIC . GranzymCOFRADIC .13CD2), or isobaric mass tags such as iTRAQ or TMT for different samples. The isotopically labelled samples are combined and digested by trypsin or another endopeptidase, e.g. GluC or lysarginase [http://flintbox.com/public/project/1948). Bound internal peptides are removed by filtration, and the unbound N-termini concentrated and analysed by LC-MS/MS. Unlike COFRADIC, TAILS does not depend on extensive fractionation and indeed performs better when analysed unfractionated, especially with the recent enhancements in MS speed and sensitivity. An aliquot of the sample prior to polymer depletion is used for a shotgun-like analysis on the same sample to normalize for differences in individual protein abundance between samples. TAILS has been successfully applied to studies on many proteases, especially members from the matrix metalloproteinase (MMP) family . Originiewed in ,71, in aiewed in  and the iewed in . TAILS hiewed in  and erytiewed in  to profiN-terminal proteomics techniques are very powerful for targeted detection of substrate cleavage by endopeptidases and aminopeptidases, but certain proteolytic events are elusive. Sometimes this is due to a redundant neo-N-terminal sequence or otherwise an unfavourable peptide for MS analysis. Cleavage by carboxypeptidases and determination of the cleavage site after release of membrane-anchored proteins from the cell surface  often mediated by the A disintegrin and metalloprotease enzymes  require6.13C and 15N isotopes, which can be resolved by modern high-resolution Fourier transform mass spectrometers. Recently, an approach was developed by the Coon lab for MS1-based quantification by metabolomics labelling that increases multiplexing capacity by using isotopologues of amino acids named neutron-encoded quantification  enable concurrent identification and multiplexed quantification of proteins in different samples. Increasing the number of isobaric tag-labelled samples that can be compared in a single experiment is highly desirable. However, it has been reported that increasing multiplexing by using larger tagging molecules, as in the case of iTRAQ4 versus iTRAQ8, decreases the number of identified and quantified proteins and peptides . The dev(NeuCode ). The speveloped .3-based methods result in substantially reduced sensitivity [3 (SPS-MS3), a recently developed technique, which allows multiple peptide-specific fragments to be accumulated and increases reporter ion intensity to balance the quantitative gains of MS3 quantification with the sensitivity required for protein-wide analysis [3, it is compatible with tryptic protein digestion; in the original MS3-based strategies from Ting et al. [2 y-ions contained a TMT tag [3 for PTMs. One option is to use different fragmentation techniques instead of collision-induced dissociation (CID), for example electron transfer dissociation . ETD haion  that prics data . Knowledics data  and TopFics data ,97 will ics data , we usedics data . MALT1 i2 quantification—only SPS-MS3 was able to visualize the partially abrogated cleavage in the patient cells. Secondly, the novel substrate HOIL1 was the only significant candidate substrate, whereas none of the known substrates were observed. This demonstrates the importance of choosing the biological experimental conditions wisely; N-terminomics methods only detect cleavage fragments that are still present, and many can be rapidly degraded after cleavage, such as known MALT1 substrate RelB. Using multiplexed labelling allows for correction of this by incorporating more time points in a single experiment.There were several interesting technical observations in this study. First, the difference in cleavage between the patient and one of the family member controls was insignificant when analysing the samples by MS7.In recent years, advances in both sample pretreatment techniques and enhanced LC-MS capabilities have opened up the field of targeted large-scale probing of PTMs by proteomics. What started with comprehensive profiling studies of phosphorylation has now been enabled for ubiquitination research, where techniques such as TUBEs and K-ϵ-GG antibody enrichment allow for identification of tens of thousands ubiquitination sites. This depth of coverage has enabled researchers to identify rare perturbations in ubiquitination events such as targets of very selective E3-conjugating enzymes or DUBs under specific (patho-) physiological conditions. Like ubiquitin research, proteomics of proteases has also matured by optimization of widely used techniques such as COFRADIC and TAILS. Our investigation in MALT1-deficient patient B cells by TMT10-TAILS has shown that with current technology it is possible to identify new key substrates of selective proteases and uncover new biological pathways. With the increasing availability of these powerful techniques, reagents and mass spectrometers, the possibility has arisen to probe multiple modifications in a comprehensive manner and truly paint a landscape of PTM in healthy and perturbed systems. Knowledge about pathways in adaptive immunity already show significant cross-talk between different PTMs, so these new technologies will undoubtedly lead to great new insights in the coming years.