Proteomics of prostate cancer serum and plasma using low and high throughput approaches.

Despite progress, MS-based proteomics in biofluids, especially blood, faces challenges such as dynamic range and throughput limitations in biomarker and disease studies. In this work, we used cutting-edge proteomics technologies to construct label-based and label-free workflows, capable of quantifying approximately 2,000 proteins in biofluids. With 70µL of blood and a single depletion strategy, we conducted an analysis of a homogenous cohort (n = 32), comparing medium-grade prostate cancer patients (Gleason score: 7(3 + 4); TNM stage: T2cN0M0, stage IIB) to healthy donors. The results revealed dozens of differentially expressed proteins in both plasma and serum. We identified the upregulation of Prostate Specific Antigen (PSA), a well-known biomarker for prostate cancer, in the serum of cancer cohort. Further bioinformatics analysis highlighted noteworthy proteins which appear to be differentially secreted into the bloodstream, making them good candidates for further exploration.

Optimal experimental design for efficient toxicity testing in microphysiological systems: A bone marrow application.

Introduction: Microphysiological systems (MPS; organ-on-a-chip) aim to recapitulate the 3D organ microenvironment and improve clinical predictivity relative to previous approaches. Though MPS studies provide great promise to explore treatment options in a multifactorial manner, they are often very complex. It is therefore important to assess and manage technical confounding factors, to maximise power, efficiency and scalability. Methods: As an illustration of how MPS studies can benefit from a systematic evaluation of confounders, we developed an experimental design approach for a bone marrow (BM) MPS and tested it for a specified context of use, the assessment of lineage-specific toxicity. Results: We demonstrated the accuracy of our multicolour flow cytometry set-up to determine cell type and maturity, and the viability of a "repeated measures" design where we sample from chips repeatedly for increased scalability and robustness. Importantly, we demonstrated an optimal way to arrange technical confounders. Accounting for these confounders in a mixed-model analysis pipeline increased power, which meant that the expected lineage-specific toxicities following treatment with olaparib or carboplatin were detected earlier and at lower doses. Furthermore, we performed a sample size analysis to estimate the appropriate number of replicates required for different effect sizes. This experimental design-based approach will generalise to other MPS set-ups. Discussion: This design of experiments approach has established a groundwork for a reliable and reproducible in vitro analysis of BM toxicity in a MPS, and the lineage-specific toxicity data demonstrate the utility of this model for BM toxicity assessment. Toxicity data demonstrate the utility of this model for BM toxicity assessment.

High-throughput mechanistic screening of non-equilibrium inhibitors by a fully automated data analysis pipeline in early drug-discovery.

Recent efforts for increasing the success in drug discovery focus on an early, massive, and routine mechanistic and/or kinetic characterization of drug-target engagement as part of a design-make-test-analyze strategy. From an experimental perspective, many mechanistic assays can be translated into a scalable format on automation platforms and thereby enable routine characterization of hundreds or thousands of compounds. However, now the limiting factor to achieve such in-depth characterization at high-throughput becomes the quality-driven data analysis, the sheer scale of which outweighs the time available to the scientific staff of most labs. Therefore, automated analytical workflows are needed to enable such experimental scale-up. We have implemented such a fully automated workflow in Genedata Screener for time-dependent ligand-target binding analysis to characterize non-equilibrium inhibitors. The workflow automates Quality Control (QC) / data modelling and decision-making process in a staged analysis: (1) quality control of raw input data-fluorescence signal-based progress curves - featuring automated rejection of unsuitable measurements; (2) automated model selection - one-step versus two-step binding model - using statistical methods and biological validity rules; (3) result visualization in specific plots and annotated result tables, enabling the scientist to review large result sets efficiently and, at the same time, to rapidly identify and focus on interesting or unusual results; (4) an interactive user interface for immediate adjustment of automated decisions, where necessary. Applying this workflow to first-pass, high-throughput kinetic studies on kinase projects has allowed us to surmount previously rate-limiting manual analysis steps and boost productivity; and is now routinely embedded in a biopharma discovery research process.

Clinicians' Experiences of Instrumented Gait Analysis in Management of Patients with Cerebral Palsy: A Qualitative Study.

AIM: To identify the interaction of instrumented gait analysis (IGA) training, expertise, and application in gait-related management of cerebral palsy. METHODS: Semi-structured interviews with 20 purposively sampled clinicians with varying professional backgrounds, expertise, and training, analyzed using the framework method. RESULTS: Fifteen sub-themes were identified within three domains: training, equipment/outputs, and roles/reasons under the core theme IGA practice. Findings were illustrated using the Experience/Equipment/Roles/Training (Exp-ERT) Framework which identifies four user categories - based on influencing factors, beset by barriers, with experience reported as a common enabling factor. Clinicians who encountered barriers in one of the domains were categorized as either "frustrated" or "hesitant" users. Those who were no longer using IGA for clinical decisions were designated "confident non-users". Finally, the 'confident experts' reported the required level of training and access to interpret IGA outputs for clinical decision-making. Expertise gained at any level of clinical practice was shown to initiate advancement within domains. CONCLUSIONS: Clinicians encounter a multitude of barriers to IGA practice that can result in failure to progress or impact on clinical decision-making. The Exp-ERT Framework emerges strongly from the data and could serve as an evaluation tool to diagnose barriers to confident expertise and support IGA-related professional development planning.

Flexible Fitting of PROTAC Concentration-Response Curves with Changepoint Gaussian Processes.

A proteolysis-targeting chimera (PROTAC) is a new technology that marks proteins for degradation in a highly specific manner. During screening, PROTAC compounds are tested in concentration-response (CR) assays to determine their potency, and parameters such as the half-maximal degradation concentration (DC50) are estimated from the fitted CR curves. These parameters are used to rank compounds, with lower DC50 values indicating greater potency. However, PROTAC data often exhibit biphasic and polyphasic relationships, making standard sigmoidal CR models inappropriate. A common solution includes manual omitting of points (the so-called masking step), allowing standard models to be used on the reduced data sets. Due to its manual and subjective nature, masking becomes a costly and nonreproducible procedure. We therefore used a Bayesian changepoint Gaussian processes model that can flexibly fit both nonsigmoidal and sigmoidal CR curves without user input. Parameters such as the DC50, maximum effect Dmax, and point of departure (PoD) are estimated from the fitted curves. We then rank compounds based on one or more parameters and propagate the parameter uncertainty into the rankings, enabling us to confidently state if one compound is better than another. Hence, we used a flexible and automated procedure for PROTAC screening experiments. By minimizing subjective decisions, our approach reduces time and cost and ensures reproducibility of the compound-ranking procedure. The code and data are provided on GitHub (https://github.com/elizavetasemenova/gp_concentration_response).

Frequency-Domain Optical Coherence Tomography for Intracranial Atherosclerotic Stenosis: Feasibility, Safety, and Preliminary Experience.

Background: Despite advances in non-invasive imaging, the characterization of atherosclerotic plaque remains superior with frequency-domain optical coherence tomography (FD-OCT) in the clinical coronary and experimental cerebrovascular literature. An assessment of the feasibility and safety of FD-OCT for intracranial atherosclerotic stenosis (ICAS) is desirable. Methods: We analyzed a cohort of all consecutive FD-OCT evaluations for ICAS performed at our institution from April 2017 to August 2018 (16 months) in patients who suffered from transient ischemic attack (TIA) or non-disabling stroke despite optimal medical management within 90 days of admission attributable to angiographically verified 70-99% stenosis of an intracranial artery. Results: Thirty-three patients harboring 36 lesions with an average age of (57.6 ± 7.1) years (male sex 27 cases) comprising nine cases of lesions located within the anterior circulation and 24 cases within the posterior circulation were identified. Of the 33 patients with 36 lesions, the FD-OCT imaging catheter detected 35/36 (97%) lesions except in one case in which the FD-OCT catheter failed to navigate excessively tortuous vessels, and FD-OCT images in 27 patients (81.8%) were finally obtained successful, where the target lesion was fully visible, and image quality under at least one pullback was graded 2 or 3. There were no symptomatic complications. Blood flow was the most common artifact encountered (51.9%). Conclusion: FD-OCT is safe and feasible for the assessment of ICAS in the anterior and posterior circulation. The use of diagnostic interferometry will have to be weighed against its cost, and these preliminary findings should be verified by prospective large-scale studies.

A multi-batch design to deliver robust estimates of efficacy and reduce animal use - a syngeneic tumour case study.

Phenotypic plasticity, the ability of a living organism to respond to the environment, can lead to conclusions from experiments that are idiosyncratic to a particular environment. The level of environmental responsiveness can result in difficulties in reproducing studies from the same institute with the same standardised environment. Here we present a multi-batch approach to in-vivo studies to improve replicability of the results for a defined environment. These multi-batch experiments consist of small independent mini-experiments where the data are combined in an integrated data analysis to appropriately assess the treatment effect after accounting for the structure in the data. We demonstrate the method on two case studies with syngeneic tumour models which are challenging due to high variability both within and between studies. Through simulations and discussions, we explore several data analysis options and the optimum design that balances practical constraints of working with animals versus sensitivity and replicability. Through the increased confidence from the multi-batch design, we reduce the need to replicate the experiment, which can reduce the total number of animals used.

Medial and Lateral Patellofemoral Joint Retinaculum Thickness in People With Patellofemoral Pain: A Case-Control Study.

OBJECTIVES: To measure the medial and lateral retinaculum thickness in individuals with and without patellofemoral pain using ultrasound and to assess associations with the symptom duration and function. METHODS: Medial and lateral patellofemoral joint retinaculum thicknesses of 32 knees (16 with patellofemoral pain and 16 asymptomatic) were measured with B-mode ultrasound at 0.5, 1, and 1.5 cm from the patella border. Participants with patellofemoral pain completed a Kujala questionnaire, and both groups underwent a single-leg squat performance assessment. Two-way analyses of variance (site × group) determined the overall effect, and Cohen d values were calculated to describe the magnitude of the difference for each measurement. RESULTS: The groups were matched for age, height, and weight. Compared to controls, participants with patellofemoral pain had thicker lateral (overall effect, P = .03) and medial (overall effect, P < 0.01) retinacula. No correlations between retinaculum thickness and Kujala scores (lateral retinaculum, r = 0.106 [0.5 cm], -0.093 [1 cm], and -0.207 [1.5 cm]; and medial retinaculum, r = 0.059, 0.109, and -0.219), symptom duration (lateral retinaculum, r = 0.001, -0.041, and 0.302; and medial retinaculum, r = -0.027, -0.358, and -0.346), or single-leg squat performance scores (lateral retinaculum, r = 0.051, 0.114, and 0.046; and medial retinaculum, r = -0.119, -0.292, and 0.011) were observed. CONCLUSIONS: Increased lateral and medial retinaculum thickness in individuals with patellofemoral pain compared to controls identifies structural changes that may be associated with the pathogenesis of patellofemoral pain. The absence of a significant correlation between retinaculum thickness and the symptom duration or function further shows a lack of an association between structure and function in individuals with patellofemoral pain.

Genome sequence of the progenitor of wheat A subgenome Triticum urartu.

Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid (Triticum turgidum, AABB) and hexaploid (Triticum aestivum, AABBDD) wheat1,2. Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping4,5. We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147 T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement.

Testing Gait with Ankle-Foot Orthoses in Children with Cerebral Palsy by Using Functional Mixed-Effects Analysis of Variance.

Existing statistical methods extract insufficient information from 3-dimensional gait data, rendering clinical interpretation of impaired movement patterns sub-optimal. We propose an alternative approach based on functional data analysis that may be worthy of exploration. We apply this to gait data analysis using repeated-measurements data from children with cerebral palsy who had been prescribed fixed ankle-foot orthoses as an example. We analyze entire gait curves by means of a new functional F test with comparison to multiple pointwise F tests and also to the traditional method - univariate repeated-measurements analysis of variance of joint angle minima and maxima. The new test maintains the nominal significance level and can be adapted to test hypotheses for specific phases of the gait cycle. The main findings indicate that ankle-foot orthoses exert significant effects on coronal and sagittal plane ankle rotation; and both sagittal and horizontal plane foot rotation. The functional F test provided further information for the stance and swing phases. Differences between the results of the different statistical approaches are discussed, concluding that the novel method has potential utility and is worthy of validation through larger scale patient and clinician engagement to determine whether it is preferable to the traditional approach.

Draft genome of the wheat A-genome progenitor Triticum urartu.

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m)A(m)), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Blockade of adhesion molecule CD146 causes pregnancy failure in mice.

Unexplained pregnancy loss and recurrent miscarriage seriously impair human fecundity. However, the underlying molecular mechanisms remain elusive. Recent studies suggest that the adhesion molecule CD146 may be involved in unexplained recurrent miscarriage. Here, we investigate the effect of CD146 on early pregnancy. Using in situ hybridization and immunohistochemistry, we found that CD146 was specifically expressed in the receptive maternal uteri and invasive embryonic trophoblasts during the early stages of pregnancy, but it was completely absent in the non-pregnant uterus. Our in vitro studies demonstrated that blocking CD146 with a function-perturbation antibody AA98 significantly inhibited the attachment of blastocysts onto the receptive uterine luminal epithelial monolayer, the trophoblastic outgrowth of blastocysts and ectoplacental cones, and the secretion of matrix metalloproteinases. Animal experiments showed that applying this antibody before embryo implantation caused pregnancy failure in mice. Our data present direct evidence for the role of CD146 in mediating embryonic attachment and trophoblastic invasion, and provide new insight into the molecular mechanism underlying unexplained pregnancy loss and recurrent miscarriage.

[A pilot study on the significance of leucocyte CD146 expression in vasculitis].

OBJECTIVE: To investigate the correlation between CD(146) expression on peripheral leucocytes with clinical activation in patients and vasculitis. METHODS: Flow cytometry was used to detect CD(146) expression on peripheral leucocytes of 39 patients with active systemic vasculitis [13 with microscopic polyangiitis (MPA), 9 with Wegener's granulomatosis (WG), 2 with chung-strauss syndrome (CSS), 9 with takayasu arteritis (TA), 2 with polyarteritis nodosa (PAN), 4 with Behcet's disease (BD)] and 24 with SLE. 18 patients (5 MPA, 4 WG, 2 CSS, 4 SLE, 2 PAN, 1 TA) were evaluated during active and improved phase. RESULTS: Expression of CD(146) on polymorphonuclears (PMN), lymphocytes (LYM) and monocytes was elevated predominantly in active vasculitis patients, especially on PMN. PMN-CD(146) positive ratio in MPA group was (20.5 +/- 25.0)%, WG (16.5 +/- 20.4)%, CSS (33.5 +/- 41.8)%, SLE (16.2 +/- 23.9)%, PAN (42.7 +/- 15.3)% and TA (6.5 +/- 4.3)%, all with significant difference (P < 0.05). BD group showed no difference with normal people (P > 0.05). LYM-CD(146) positive ratio in MPA group was (7.3 +/- 8.4)%, WG (9.6 +/- 9.1)%, CSS (16.1 +/- 20.1)%, SLE (12.2 +/- 13.5)% and TA (5.1 +/- 4.4)%, all with significant difference (P < 0.05). BD and PAN group showed no difference (P > 0.05). The expression of CD(146) on the three groups of cells correlated with each other (r was 0.66 and 0.853 respectively, P = 0.000), but they did not correlate with the course of the disease, age, ESR, C reactive protein (CRP), antineutrophil cytoplasmic antibody (ANCA), birmingham vasculitis activity score (BVAS) and SLE disease activity index (SLEDAI) (P > 0.5). 18 patients were followed up after therapy with corticosteroids and immunosuppressive drugs. PMN-CD(146) positive ratio during active phase and improved phase was (40.3 +/- 27.0)% and (9.9 +/- 18.8)% (P = 0.001) and LYM-CD(146) positive ratio was (15.7 +/- 13.6)% and (6.7 +/- 5.3)% (P = 0.021) respectively. Almost in all of them the ratio decreased from active phase to improved phase. These were first reported in the world literature. CONCLUSION: A high expression of CD(146) on peripheral leucocytes especially PMN in patients with active vasculitis is reported here for the first time and it turns to be negative with the therapy of corticosteroid and immunosuppressive drugs. This indicates that CD(146) may be a new marker for disease activation and may be important in the pathogenesis of vasculitis. Further study in this respect in needed.