QuantifyPolarity, a new tool-kit for measuring planar polarized protein distributions and cell properties in developing tissues.

The coordination of cells or structures within the plane of a tissue is known as planar polarization. It is often governed by the asymmetric distribution of planar polarity proteins within cells. A number of quantitative methods have been developed to provide a readout of planar polarized protein distributions. However, previous planar polarity quantification methods can be affected by variation in cell geometry. Hence, we developed a novel planar polarity quantification method based on Principal Component Analysis (PCA) that is shape insensitive. Here, we compare this method with other state-of-the-art methods on simulated models and biological datasets. We found that the PCA method performs robustly in quantifying planar polarity independently of variation in cell geometry and other image conditions. We designed a user-friendly graphical user interface called QuantifyPolarity, equipped with three polarity methods for automated quantification of polarity. QuantifyPolarity also provides tools to quantify cell morphology and packing geometry, allowing the relationship of these characteristics to planar polarization to be investigated. This tool enables experimentalists with no prior computational expertise to perform high-throughput cell polarity and shape analysis automatically and efficiently.

Cr5.7Si2.3P8N24-A Chromium(+IV) Nitridosilicate Phosphate with Amphibole-Type Structure.

The first nitridic analog of an amphibole mineral, the quaternary nitridosilicate phosphate Cr5.7Si2.3P8N24 was synthesized under high-pressure high-temperature conditions at 1400 °C and 12 GPa from the binary nitrides Cr2N, Si3N4 and P3N5, using NH4N3 and NH4F as additional nitrogen source and mineralizing agent, respectively. The crystal structure was elucidated by single-crystal X-ray diffraction with microfocused synchrotron radiation (C2/m, a=9.6002(19), b=17.107(3), c=4.8530(10) Å, ß=109.65(3)°). The elemental composition was analyzed by energy dispersive X-ray spectroscopy. The structure consists of vertex-sharing PN4-tetrahedra forming zweier double chains and edge-sharing (Si,Cr)-centered octahedra forming separated ribbons. Atomic resolution scanning transmission electron microscopy shows ordered Si and Cr sites next to a disordered Si/Cr site. Optical spectroscopy indicates a band gap of 2.1 eV. Susceptibility measurements show paramagnetic behavior and support the oxidation state Cr+IV, which is confirmed by EPR. The comprehensive analysis expands the field of Cr-N chemistry and provides access to a nitride analog of one of the most prevalent silicate structures.

A theoretical framework for planar polarity establishment through interpretation of graded cues by molecular bridges.

Planar polarity is a widespread phenomenon found in many tissues, allowing cells to coordinate morphogenetic movements and function. A common feature of animal planar polarity systems is the formation of molecular bridges between cells, which become polarised along a tissue axis. We propose that these bridges provide a general mechanism by which cells interpret different forms of tissue gradients to coordinate directional information. We illustrate this using a generalised and consistent modelling framework, providing a conceptual basis for understanding how different mechanisms of gradient function can generate planar polarity. We make testable predictions of how different gradient mechanisms can influence polarity direction.

Metadata Framework to Support Deployment of Digital Health Technologies in Clinical Trials in Parkinson's Disease.

Sensor data from digital health technologies (DHTs) used in clinical trials provides a valuable source of information, because of the possibility to combine datasets from different studies, to combine it with other data types, and to reuse it multiple times for various purposes. To date, there exist no standards for capturing or storing DHT biosensor data applicable across modalities and disease areas, and which can also capture the clinical trial and environment-specific aspects, so-called metadata. In this perspectives paper, we propose a metadata framework that divides the DHT metadata into metadata that is independent of the therapeutic area or clinical trial design (concept of interest and context of use), and metadata that is dependent on these factors. We demonstrate how this framework can be applied to data collected with different types of DHTs deployed in the WATCH-PD clinical study of Parkinson's disease. This framework provides a means to pre-specify and therefore standardize aspects of the use of DHTs, promoting comparability of DHTs across future studies.

Pyrroloquinoline Quinone Aza-Crown Ether Complexes as Biomimetics for Lanthanide and Calcium Dependent Alcohol Dehydrogenases*.

Understanding the role of metal ions in biology can lead to the development of new catalysts for several industrially important transformations. Lanthanides are the most recent group of metal ions that have been shown to be important in biology, that is, in quinone-dependent methanol dehydrogenases (MDH). Here we evaluate a literature-known pyrroloquinoline quinone (PQQ) and 1-aza-15-crown-5 based ligand platform as scaffold for Ca2+ , Ba2+ , La3+ and Lu3+ biomimetics of MDH and we evaluate the importance of ligand design, charge, size, counterions and base for the alcohol oxidation reaction using NMR spectroscopy. In addition, we report a new straightforward synthetic route (3 steps instead of 11 and 33 % instead of 0.6 % yield) for biomimetic ligands based on PQQ. We show that when studying biomimetics for MDH, larger metal ions and those with lower charge in this case promote the dehydrogenation reaction more effectively and that this is likely an effect of the ligand design which must be considered when studying biomimetics. To gain more information on the structures and impact of counterions of the complexes, we performed collision induced dissociation (CID) experiments and observe that the nitrates are more tightly bound than the triflates. To resolve the structure of the complexes in the gas phase we combined DFT-calculations and ion mobility measurements (IMS). Furthermore, we characterized the obtained complexes and reaction mixtures using Electron Paramagnetic Resonance (EPR) spectroscopy and show the presence of a small amount of quinone-based radical.

Mesoionic Carbenes in Low- to High-Valent Vanadium Chemistry.

We report the synthesis of vanadium(V) oxo complex 1 with a pincer-type dianionic mesoionic carbene (MIC) ligand L1 and the general formula [VOCl(L1)]. A comparison of the structural (SC-XRD), electronic (UV-vis), and electrochemical (cyclic voltammetry) properties of 1 with the benzimidazolinylidene congener 2 (general formula [VOCl(L2)]) shows that the MIC is a stronger donor also for early transition metals with low d-electron population. Since electrochemical studies revealed both complexes to be reversibly reduced, the stronger donor character of MICs was not only demonstrated for the vanadium(V) but also for the vanadium(IV) oxidation state by isolating the reduced vanadium(IV) complexes [Co(Cp*)2][1] and [Co(Cp*)2][2] ([Co(Cp*)2] = decamethylcobaltocenium). The electronic structures of the compounds were investigated by computational methods. Complex 1 was found to be a moderate precursor for salt metathesis reactions, showing selective reactivity toward phenolates or secondary amides, but not toward primary amides and phosphides, thiophenols, or aryls/alkyls donors. Deoxygenation with electron-rich phosphines failed to give the desired vanadium(III) complex. However, treatment of the deprotonated ligand precursor with vanadium(III) trichloride resulted in the clean formation of the corresponding MIC vanadium(III) complex 6, which undergoes a clean two-electron oxidation with organic azides yielding the corresponding imido complexes. The reaction with TMS-N3 did not afford a nitrido complex, but instead the imido complex 10. This study reveals that, contrary to popular belief, MICs are capable of supporting early transition-metal complexes in a variety of oxidation states, thus making them promising candidates for the activation of small molecules and redox catalysis.

Library consultations and a global pandemic: An analysis of consultation difficulty during COVID-19 across multiple factors.

The purpose of this study is to examine the relationship between librarians' perception of the difficulty of patron consultations and a variety of factors that characterize these interactions in the context of an academic library at a large public university. The study also provides insight into how changes in library service operations due to the global COVID-19 pandemic have affected the perceived difficulty of library consultations. Data samples were drawn from a LibInsight dataset and limited to consultations from Fall 2019 and Spring 2020 (N = 3331). Statistical analysis was conducted using ordinal logistic regression to quantify the relationship between perceptions of difficulty and factors indicating pre/post-COVID-19 modifications, patron type, scheduling, question format, library department, consultation duration, semester, and campus. Most notably, results indicate a statistically significant (p < 0.001) increase in the perceived difficulty of consultations that followed the closure of the library's physical spaces due to COVID-19, even when controlling for other factors in multiple model formulations. These results, as well as insights pertaining to other factors associated with library consultations and perceptions of difficulty, have implications for how librarians frame, understand, and manage their workloads. Additionally, findings may provide library service managers with the evidence needed to better coordinate and evaluate library services.

A genome-wide RNAi screen identifies MASK as a positive regulator of cytokine receptor stability.

Cytokine receptors often act via the Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway to form a signalling cascade that is essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, and their activity and dimerisation, we analysed the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. We show that MASK is able to regulate receptor levels and JAK/STAT signalling both in vitro and in vivo We also show that its human homologue, ANKHD1, is also able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, our results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health.This article has an associated First Person interview with the first author of the paper.

Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment.

The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10-14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

Collaboration between experiment and theory in solar fuels research.

As the challenges in science increase in scope and interdisciplinarity, collaboration becomes increasingly important. Our groups have maintained close collaborations for solar fuels research over the past decade. Based on this experience, we discuss strategies for collaboration between experiment and theory including facilitation of effective communication and navigation of problems that arise. These strategies are illustrated by case studies of collaborative efforts in solar fuels research pertaining to interfacial electron transfer in dye-sensitized metal oxides and the design and mechanism of water-oxidation catalysts.

A Pyridine Alkoxide Chelate Ligand That Promotes Both Unusually High Oxidation States and Water-Oxidation Catalysis.

Water-oxidation catalysis is a critical bottleneck in the direct generation of solar fuels by artificial photosynthesis. Catalytic oxidation of difficult substrates such as water requires harsh conditions, so the ligand must be designed both to stabilize high oxidation states of the metal center and to strenuously resist ligand degradation. Typical ligand choices either lack sufficient electron donor power or fail to stand up to the oxidizing conditions. Our research on Ir-based water-oxidation catalysts (WOCs) has led us to identify a ligand, 2-(2'-pyridyl)-2-propanoate or "pyalk", that fulfills these requirements. Work with a family of Cp*Ir(chelate)Cl complexes had indicated that the pyalk-containing precursor gave the most robust WOC, which was still molecular in nature but lost the Cp* fragment by oxidative degradation. In trying to characterize the resulting active "blue solution" WOC, we were able to identify a diiridium(IV)-mono-µ-oxo core but were stymied by the extensive geometrical isomerism and coordinative variability. By moving to a family of monomeric complexes [IrIII/IV(pyalk)3] and [IrIII/IV(pyalk)2Cl2], we were able to better understand the original WOC and identify the special properties of the ligand. In this Account, we cover some results using the pyalk ligand and indicate the main features that make it particularly suitable as a ligand for oxidation catalysis. The alkoxide group of pyalk allows for proton-coupled electron transfer (PCET) and its strong σ- and π-donor power strongly favors attainment of exceptionally high oxidation states. The aromatic pyridine ring with its methyl-protected benzylic position provides strong binding and degradation resistance during catalytic turnover. Furthermore, the ligand has two additional benefits: broad solubility in aqueous and nonaqueous solvents and an anisotropic ligand field that enhances the geometry-dependent redox properties of its complexes. After discussion of the general properties, we highlight the specific complexes studied in more detail. In the iridium work, the isolated mononuclear complexes showed easily accessible Ir(III/IV) redox couples, in some cases with the Ir(IV) state being indefinitely stable in water. We were able to rationalize the unusual geometry-dependent redox properties of the various isomers on the basis of ligand-field effects. Even more striking was the isolation and full characterization of a stable Rh(IV) state, for which prior examples were very reactive and poorly characterized. Importantly, we were able to convert monomeric Ir complexes to [Cl(pyalk)2IrIV-O-IrIVCl(pyalk)2] derivatives that help model the "blue solution" properties and provide groundwork for rational synthesis of active, well-defined WOCs. More recent work has moved toward the study of first-row transition metal complexes. Manganese-based studies have highlighted the importance of the chelate effect for labile metals, leading to the synthesis of pincer-type pyalk derivatives. Beyond water oxidation, we believe the pyalk ligand and its derivatives will also prove useful in other oxidative transformations.

Effect of methotrexate on JAK/STAT pathway activation in myeloproliferative neoplasms.

BACKGROUND: The myeloproliferative neoplasms are a group of haematological malignancies characterised by pathological activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) intracellular signalling pathway. 50-95% of patients have an acquired mutation (JAK2V617F) causing constitutive activation of JAK2. Our aim was to find new treatments for myeloproliferative neoplasms by identifying compounds that suppress JAK/STAT pathway activation. METHODS: We used a luciferase-based transcriptional assay in the low complexity Drosophila model system to screen a library of 2000 small molecules for modulators of JAK/STAT pathway activation. Screen hits were validated with western blotting in the HDLM-2 Hodgkin's lymphoma cell line. The HEL cell line, in which constitutive JAK/STAT pathway activation is caused by JAK2V617F, was used to determine the relevance of screen hits for treatment of myeloproliferative neoplasms. FINDINGS: Methotrexate and the chemically similar drug aminopterin were independently identified as strong inhibitors of the Drosophila JAK/STAT pathway, an effect conserved to human cells. Methotrexate did not affect protein phosphorylation in other intracellular signalling pathways. Methotrexate caused significant suppression of JAK/STAT activation in HEL cells at a concentration equivalent to that seen in patients taking low-dose oral methotrexate (p≤0·001). INTERPRETATION: Our results suggest that methotrexate is a promising treatment for myeloproliferative neoplasms that could be translated into clinical trials after assessment in primary cells. These results are particularly relevant in myelofibrosis. Inhibitors of JAK1/2 improve symptoms and prolong life in myelofibrosis, but their use is limited by cost. Other existing therapies for myelofibrosis appear no more effective than placebo. Methotrexate might bring the benefits of JAK/STAT pathway inhibition at a lower cost. FUNDING: Cancer Research UK, Yorkshire Cancer Research, UK Medical Research Council, Wellcome Trust, EU Framework Cancer Pathways.

Visualization and quantitation of spine deformity in zebrafish models of scoliosis by micro-computed tomography.

Zebrafish (Danio rerio) are increasingly used to investigate spine development, growth, and for studying the etiology of spinal deformity, such as scoliosis. Here, we present a micro-computed tomography-based pipeline for visualizing the zebrafish skeleton. We describe steps for sample preparation, imaging, data management, and processing. We then detail analysis of vertebral and spine morphology using open-source software. This protocol will be useful for scientists using zebrafish to understand spine development and disease. For complete details on the use and execution of this protocol, please refer to Bearce et al. (2022).1.

Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway.

BACKGROUND: Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. RESULTS: Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. CONCLUSIONS: Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.

The 5-hydroxytryptamine4 receptor agonists prucalopride and PRX-03140 increase acetylcholine and histamine levels in the rat prefrontal cortex and the power of stimulated hippocampal θ oscillations.

5-Hydroxytryptamine (5-HT)(4) receptor agonists reportedly stimulate brain acetylcholine (ACh) release, a property that might provide a new pharmacological approach for treating cognitive deficits associated with Alzheimer's disease. The purpose of this study was to compare the binding affinities, functional activities, and effects on neuropharmacological responses associated with cognition of two highly selective 5-HT(4) receptor agonists, prucalopride and 6,7-dihydro-4-hydroxy-7-isopropyl-6-oxo-N-[3-(piperidin-1-yl)propyl]thieno[2,3-b]pyridine-5-carboxamide (PRX-03140). In vitro, prucalopride and PRX-03140 bound to native rat brain 5-HT(4) receptors with K(i) values of 30 nM and 110 nM, respectively, and increased cAMP production in human embryonic kidney-293 cells expressing recombinant rat 5-HT(4) receptors. In vivo receptor occupancy studies established that prucalopride and PRX-03140 were able to penetrate the brain and bound to 5-HT(4) receptors in rat brain, achieving 50% receptor occupancy at free brain exposures of 330 nM and 130 nM, respectively. Rat microdialysis studies revealed that prucalopride maximally increased ACh and histamine levels in the prefrontal cortex at 5 and 10 mg/kg, whereas PRX-03140 significantly increased cortical histamine levels at 50 mg/kg, failing to affect ACh release at doses lower than 150 mg/kg. In combination studies, donepezil-induced increases in cortical ACh levels were potentiated by prucalopride and PRX-03140. Electrophysiological studies in rats demonstrated that both compounds increased the power of brainstem-stimulated hippocampal θ oscillations at 5.6 mg/kg. These findings show for the first time that the 5-HT(4) receptor agonists prucalopride and PRX-03140 can increase cortical ACh and histamine levels, augment donepezil-induced ACh increases, and increase stimulated-hippocampal θ power, all neuropharmacological parameters consistent with potential positive effects on cognitive processes.

Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation.

Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease-related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte-like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK-induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F-mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA-derived data, quantify their role in regulating the proliferation of cancer-derived cell lines. Such an inter-species approach is an effective way to identify factors with conserved functions that might be central to human disease.

Validation of the MYChrOme™ Culture Plate for Detection and Differentiation of Rapid-Growing Nontuberculous Mycobacteria in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 062101.

BACKGROUND: The MYChrOme™ Culture Plate is a chromogenic media for the detection and differentiation of rapid-growing nontuberculous mycobacteria (NTM) in water, aided by MYCOn™ decontamination to reduce background microbiota. OBJECTIVE: Evaluate the MYChrOme Culture Plates for the detection of rapid-growing NTM in potable and non-potable water as part of the AOAC Performance Tested Method(s)SM program. METHODS: Inclusivity and exclusivity of MYChrOme were evaluated with 50 target and 30 non-target organisms. Method robustness and lot stability of MYChrOme were analyzed. The candidate method was compared to a modified US Food and Drug Administration (FDA) Method: U.S. FDA-Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks using an equivalency test. The matrix study consisted of artificially contaminated potable water and naturally contaminated non-potable water. Independent laboratory testing was conducted to verify method performance in non-potable water. RESULTS: The inclusivity of MYChrOme was 94% within one week, and 98% within two weeks. The exclusivity was 96% for untreated samples and 100% for treated samples. The candidate method remained statistically equivalent for robustness and a three-month shelf-life was confirmed. For both matrixes, the candidate and reference methods were not equivalent, with more colonies enumerated on the candidate method except for one contamination level of the potable matrix. CONCLUSION: The MYChrOme culture method can successfully detect and differentiate rapid-growing NTM in the matrixes tested, with sensitivity equivalent or higher than the reference method. HIGHLIGHTS: The MYChrOme culture plate offers differentiation of rapid-growing NTM colonies, improved detection in non-potable samples with MYCOn decontamination, and results within 7 days.

Inaccuracies of the ISO 11731 Method for Environmental Validation of Legionella in Building Water Systems: Opportunities to Improve Sensitivity and Detect Viable but Non-Culturable Legionella.

Current environmental diagnostics for the detection of Legionella fail to detect viable but non-culturable Legionella, have sensitivity limitations and are time-consuming (10-14 days to results). The objective of this study was to compare Legionella detection results between the standard ISO 11731 and an innovative Legionella detection method that utilizes a hybrid methodology of traditional microbiology and molecular detection. In this study, four hundred and seventy-six (476) potable building water samples were analyzed with ISO 11731 and the novel method in parallel. Of the 476 total samples that were tested, a discrepancy of 21% was observed when comparing the ISO 11731 method to the novel method. Separating the samples based on hazard control methods yielded a 15.4% discrepancy for chlorinated systems (n = 284) and a 29% discrepancy for monochloraminated systems (n = 192). The data presented here conclusively show inaccuracies in environmental validation for building water systems based on results returned by the standard ISO 11731 method. This is especially evident in systems primarily disinfected with monochloramines. Overall, these data highlight the need for new and innovative methods to overcome the inaccuracies of the traditional ISO 11731 spread plates to prevent disease and injury caused by Legionella.

Identifying and characterising sources of variability in digital outcome measures in Parkinson's disease.

Smartphones and wearables are widely recognised as the foundation for novel Digital Health Technologies (DHTs) for the clinical assessment of Parkinson's disease. Yet, only limited progress has been made towards their regulatory acceptability as effective drug development tools. A key barrier in achieving this goal relates to the influence of a wide range of sources of variability (SoVs) introduced by measurement processes incorporating DHTs, on their ability to detect relevant changes to PD. This paper introduces a conceptual framework to assist clinical research teams investigating a specific Concept of Interest within a particular Context of Use, to identify, characterise, and when possible, mitigate the influence of SoVs. We illustrate how this conceptual framework can be applied in practice through specific examples, including two data-driven case studies.

A microtubule interactome: complexes with roles in cell cycle and mitosis.

The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.