Using online tools at the Bovine Genome Database to manually annotate genes in the new reference genome.

With the availability of a new highly contiguous Bos taurus reference genome assembly (ARS-UCD1.2), it is the opportune time to upgrade the bovine gene set by seeking input from researchers. Furthermore, advances in graphical genome annotation tools now make it possible for researchers to leverage sequence data generated with the latest technologies to collaboratively curate genes. For many years the Bovine Genome Database (BGD) has provided tools such as the Apollo genome annotation editor to support manual bovine gene curation. The goal of this paper is to explain the reasoning behind the decisions made in the manual gene curation process while providing examples using the existing BGD tools. We will describe the sources of gene annotation evidence provided at the BGD, including RNA-seq and Iso-Seq data. We will also explain how to interpret various data visualizations when curating gene models, and will demonstrate the value of manual gene annotation. The process described here can be applied to manual gene curation for other species with similar tools. With a better understanding of manual gene annotation, researchers will be encouraged to edit gene models and contribute to the enhancement of livestock gene sets.

Characterization and application studies of ProxyPhos, a chemosensor for the detection of proximally phosphorylated peptides and proteins in aqueous solutions.

Proximal phosphorylation on proteins appears to have functional significance and has been associated with several diseases, including Alzheimer's and cancer. While much remains to be learned about the role of proximal phosphorylation in biological systems, no simple and/or affordable technique is available for its detection. To this end, we have previously developed a ProxyPhos chemosensor, which detects proximally phosphorylated peptides and proteins over mono- and non-phosphorylated motifs in aqueous solutions. In this follow-up work, we performed extensive characterization of peptide and protein ProxyPhos assay conditions to achieve enhanced detection, and further explored the selectivity of ProxyPhos, and its potential off-targets. As a result of characterization studies, selective sensing of proximally phosphorylated over mono-phosphorylated peptides and proteins was achieved. Moreover, studies demonstrated that ProxyPhos was compatible with the detection of all commonly phosphorylated residues (i.e. tyrosine, serine and threonine residues). Under optimized conditions, ProxyPhos efficiently discriminated between peptides derived from the activated (proximally phosphorylated, disease-relevant) and inactive (mono-phosphorylated) forms of JAK2, SYK and MAPK1 kinases. In addition, ProxyPhos can be used to probe phosphatase activity on peptides and proteins via detecting changes in proximal phosphorylation, demonstrating immediate utility of this chemosensing system.

Structural insights into the ene-reductase synthesis of profens.

Reduction of double bonds of α,ß-unsaturated carboxylic acids and esters by ene-reductases remains challenging and it typically requires activation by a second electron-withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-crystal structural analysis of profen-bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-profens was established and this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-active (S)-profens.

L-cysteine suppresses ghrelin and reduces appetite in rodents and humans.

BACKGROUND: High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. METHODS: We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. RESULTS: l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. CONCLUSIONS: Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety.

Survival of HIV-positive individuals with hepatitis B and C infection in Michigan.

We sought to estimate mortality and associated factors in HIV-hepatitis co-infected individuals in Michigan using a retrospective cohort study. For the study period of 1 January 2006 to 31 December 2009, all HIV-infected individuals were matched to hepatitis B and C cases. In the final Cox proportional hazards regression model, individuals of other [hazard ratio (HR) 2·2, 95% confidence interval (CI) 1·4-3·2] and black (HR 1·3, 95% CI 1·1-1·6) race had decreased survival compared to white race. Similarly, injecting drug users (IDUs) (HR 2·1, 95% CI 1·6-2·6), men who have sex with men (MSM)/IDUs (HR 1·5, 95% CI 1·1-2·2), individuals with undetermined risk (HR 1·5, 95% CI 1·2-1·9) and heterosexual practices (HR 1·4, 95% CI 1·1-1·8) had decreased survival compared to MSM. Additionally, an interaction was found between current HIV status and co-infection. Mortality in HIV-hepatitis co-infected individuals remains a continuing problem. Our study can help in planning interventions to reduce mortality in HIV-infected individuals.

Relationship between knee pain and the presence, location, size and phenotype of femorotibial denuded areas of subchondral bone as visualized by MRI.

OBJECTIVE: Conflicting associations between imaging biomarkers and pain in knee osteoarthritis (OA) have been reported. A relation between pain and denuded areas of subchondral bone (dABs) has been suggested and this study explores this relationship further by relating the presence, phenotype, location and size of dABs to different measures of knee pain. METHODS: 633 right knees from the Osteoarthritis Initiative (OAI) (250 men, age 61.7 ± 9.6 yrs, BMI 29.4 ± 4.7 kg/m(2)) were included. Manual segmentation of the femorotibial cartilage plates was performed on 3 T coronal fast low angle shot with water excitation (FLASHwe) images. dABs were defined as areas where the subchondral bone was uncovered by cartilage. The following measures of pain were used: weightbearing-, non-weightbearing-, moderate-to-severe-, infrequent- and frequent knee pain. RESULTS: Using pain measures from subjects without dABs as a reference, those with at least one dAB had a 1.64-fold higher prevalence ratio [PR, 95% confidence interval (CI) 1.24-2.18] to have frequent and 1.45-fold higher for moderate-to-severe knee pain (95% CI 1.13-1.85). Subjects with dABs in central subregions had a 1.53-fold increased prevalence of having weightbearing pain (95% CI 1.20-1.97), especially when the central subregion was moderately (>10%) denuded (PR 1.81, 95% CI 1.35-2.42). Individuals with cartilage-loss-type dABs had a slightly higher prevalence (PR 1.13, 95% CI 1.00-1.27) of having frequent knee pain compared to individuals with intra-chondral-osteophyte-type dABs. CONCLUSION: This study supports a positive relation between femorotibial dABs and knee pain, especially when the dABs are located centrally (i.e., in weightbearing regions) or when the respective central subregion is moderately denuded.

Hepatitis B and C co-infection in HIV/AIDS population in the state of Michigan.

A retrospective cohort study was conducted from 1 January 2006 to 31 December 2009 in Michigan to estimate the prevalence of HIV and hepatitis co-infection and identify associated factors. The prevalence of co-infection was 4.1% [95% confidence interval (CI) 3.8-4.5]. Multivariable logistic regression analysis revealed a significant association between co-infection and being male and: of Black race [odds ratio (OR) 2.0, 95% CI 1.2-3.6] and of Other race (OR 3.5, 95% CI 1.7-7.0) compared to Hispanic race. A significant association was found between co-infection and risk categories of blood products (OR 11.1, 95% CI 6.2-20.2), injecting drug user (IDU) (OR 3.6, 95% CI 2.7-4.8) and men who have sex with men/IDU (OR 3.4, 95% CI 2.4-4.9) in addition to two interactions; one between sex and current HIV status and the other between current HIV status and age at HIV diagnosis. Our results document the changing epidemiology of HIV-hepatitis co-infection which can guide preventive measures and interventions to reduce the prevalence of hepatitis co-infection.

Usefulness of the Memorial Sloan Kettering Cancer Center nomogram in the prognosis of patients treated with radical cystectomy for invasive bladder cancer.

OBJECTIVES: To evaluate the usefulness of the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram for prediction of recurrence probability in our series of patients who have undergone radical cystectomy for bladder cancer. METHODS: 397 patients underwent radical cystectomy for bladder cancer between 1986 and 2005. 165 patients were excluded:21 due to exitus in the immediate postoperative period, 32 due to previous radiation therapy, 6 due to neoadjuvant chemotherapy, 5 due to inability to complete follow-up, 15 that did not undergo lymphadenectomy and 86 who were alive at the time of review with less than 5 years of follow-up. Patients were classified into recurrence risk groups: organ-confined tumors (pT0-2 pN0 ), extra-bladder involvement (pT3-4 pN0) and lymph node involvement (pN+). Survival analysis was performed using the Kaplan-Meier method. Five-year recurrence-free survival by risk groups in our series was compared with the one estimated using the MSKCC nomogram using a ROC curve. RESULTS: We analyzed 232 patients. Follow-up in patients who died of cancer was 25 ± 25 months. For alive patients and those who died of other causes, follow-up was 120 ± 39 months. Pathology studies revealed 42.7% organ-confined tumors , 33.2% with extra-bladder involvement and 24.1% with lymph node involvement. The five-year recurrence free survival analysis according to the Kaplan-Meier method stratified by risk groups was: pT0-2 76%, pT3-4 51%, pN+ 31%. The probability of recurrence free survival according to the MSKCC nomogram in the same risk groups was: 85% ± 5%, 62% ± 10% and 25% ± 13%, respectively. The area under the ROC curve was 0.795 (95% CI 0.739-0.852) CONCLUSION: In our series, the MSKCC nomogram constitutes a useful tool for predicting 5-year cancer free survival in patients who undergo radical cystectomy.

[Urachal adenocarcinoma treated with robotic assisted laparoscopy partial cystectomy]. / Cistectomóa parcial laparoscópica asistida por robot Da Vinci para el tratamiento del adenocarcinoma de uraco.

OBJECTIVE: To describe a case of urachal adenocarcinoma treated with robotic assisted laparoscopic partial cystectomy and en-bloc exeresis of urachus and umbilicus and bibliographic review. METHODS: A 63 year-old man with hematuria and hypogastric pain. He was diagnosed of urachal adenocarcinoma by transurethral resection and axial tomography. We performed a robotic assisted laparoscopic partial cystectomy using a da Vinci® S HD (Intuitive Surgical System) device. We describe the surgical technique and examine total length of time for surgery and for console, pathology report, margin status, postoperative outcome and oncological status 7 months after surgery. RESULTS: 4 ports were used for robotic arms and one additional for the assistant. Cystoscopy was performed during surgery to mark tumor margins. Bladder was closed using a running suture with Poliglactin 0. Total length time for surgery was 2hs 28 minutes, console time was 1h54'. Two days later patient was discharged and no complication was reported. After two weeks Foley cathether was removed and bladder volume was 300ml. Pathology report informed undifferentiated urachal adenocarcinoma with perivesical tissue infiltration with margins free from tumor, corresponding to Sheldon IIIB and Ontario III classification. Seven months later patient was fee from recurrence. CONCLUSION: Robotic assisted laparoscopy partial cystectomy with en-bloc exeresis of urachal and umbilicus is feasible.

BCG shortage for intravesical instillation is associated with early tumoral recurrence in patients with high-risk non-muscle invasive bladder tumours.

INTRODUCTION: During 2019 there was a worldwide shortage of BCG strains for intravesical instillation, limiting the availability of full dose schemes for maintenance courses. The main objective was to analyze the impact on tumoral relapse secondary to BCG shortage in our center. Secondary outcomes included recurrence and progression-free survival rates and tumoral relapse specific characteristics. METHODS: Retrospective cohort study including 158 subjects (64 treated during 2019 and 94 during 2017) with high-risk non-muscle invasive bladder cancer and treated with a combination of Transurethral bladder resection (TURB) followed by adjuvant intravesical instillation with BCG in a tertiary hospital in Spain. Basal characteristics of both groups were analyzed. Times to event of interest (relapse; including recurrence and/or progression) were estimated with Kaplan-Meier survival analysis. Disease-free survival rates were analyzed using a multivariable Cox regression model of proportional hazards. RESULTS: Median follow-up in the 2019 sample was 24 months and 50 months in the 2017 group with a median number of instillations of 8 and 12 respectively. Median time to relapse of 285 days (145-448) during 2019 and 382 days (215-567) in 2017 were observed (logRank p = 0.025). Further multivariable analysis revealed a proportional hazard ratio (HR) for disease-free survival rate of 1.87 (95% CI: 1.04-3.37 p = 0.036). No statistically significant differences in tumoral relapse characteristics were observed. CONCLUSION: BCG shortage and subsequent reduced-dose schemes used for intravesical instillation due to limited availability, increase early tumoral relapse rates. These findings are consistent with available evidence, showing the need for full-dose BCG courses.

Methodologic issues in clinical trials for prevention or risk reduction in osteoarthritis.

The design and execution of prevention trials for OA have methodological issues that are distinct from trials designed to impact prevalent disease. Disease definitions and their precise and sensitive measurement, identification of high-risk populations, the nature of the intervention (pharmaceutical, nutraceutical, behavioral) and its potential pleiotropic impacts on other organ systems are critical to consider. Because prevention trials may be prolonged, close attention to concomitant life changes and co-morbidities, adherence and participant retention in the trial is of primary importance, as is recognition of the potential for "preventive misconception" and "behavioral disinhibition" to affect the ability of the trial to show an effect of the intervention under study. None of these potential pitfalls precludes a successful and scientifically rigorous process and outcome. As technology improves the means to measure and predict the OA process and its clinical consequences, it will be increasingly possible to screen individuals for high-risk phenotypes, combining clinical factors with information from imaging, genetic, metabolic and other biomarkers and to impact this high-risk condition to avoid or delay OA both structurally and symptomatically.

Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. METHODS: The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). RESULTS: This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. CONCLUSIONS: Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1-2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.

Palladin isoforms 3 and 4 regulate cancer-associated fibroblast pro-tumor functions in pancreatic ductal adenocarcinoma.

Pancreatic Ductal Adenocarcinoma (PDAC) has a five-year survival under 10%. Treatment is compromised due to a fibrotic-like stromal remodeling process, known as desmoplasia, which limits therapeutic perfusion, supports tumor progression, and establishes an immunosuppressive microenvironment. These processes are driven by cancer-associated fibroblasts (CAFs), functionally activated through transforming growth factor beta1 (TGFß1). CAFs produce a topographically aligned extracellular matrix (ECM) that correlates with reduced overall survival. Paradoxically, ablation of CAF populations results in a more aggressive disease, suggesting CAFs can also restrain PDAC progression. Thus, unraveling the mechanism(s) underlying CAF functions could lead to therapies that reinstate the tumor-suppressive features of the pancreatic stroma. CAF activation involves the f-actin organizing protein palladin. CAFs express two palladin isoforms (iso3 and iso4) which are up-regulated in response to TGFß1. However, the roles of iso3 and iso4 in CAF functions remain elusive. Using a CAF-derived ECM model, we uncovered that iso3/iso4 are required to sustain TGFß1-dependent CAF activation, secrete immunosuppressive cytokines, and produce a pro-tumoral ECM. Findings demonstrate a novel role for CAF palladin and suggest that iso3/iso4 regulate both redundant and specific tumor-supportive desmoplastic functions. This study highlights the therapeutic potential of targeting CAFs to restore fibroblastic anti-tumor activity in the pancreatic microenvironment.

Sonation in the male common snipe (Capella gallinago gallinago L.) is achieved by a flag-like fluttering of their tail feathers and consequent vortex shedding.

Male common snipe (Capella gallinago gallinago) produce a 'drumming' sound with their outer tail feathers during their mating dives, but little is known about how this is achieved. We investigated the movements and sound producing capabilities of the outer tail feathers. Using a wind tunnel, we compared observations of the frequencies of sound produced with the predictions from aerodynamic theory. The feathers were also filmed in an air-flow with a high speed video camera, and subjected to morphological examination and biomechanical testing. We propose a mechanistic hypothesis of how the modified outer feathers of the male common snipe generate sound, and the adaptations that facilitate this. Video and audio analysis of the feather demonstrated that a fluttering of the trailing vane generated the sound. The flutter of the vane is facilitated by the rearward curvature of the feather shaft, reduced branching angles of the barbs in the trailing vane and the lack of hooks on the barbs along a hinge region, all of which increase its flexural compliance. Sound production occurred at the same frequency as the vane movements, at frequencies consistent with it being produced by a fluttering flag mechanism powered by vortex shedding.

Central and peripheral administration of human relaxin-2 to adult male rats inhibits food intake.

AIM: Relaxin is a polypeptide hormone involved in pregnancy and lactation. It is mainly secreted by the corpus luteum and placenta, but is expressed in a number of other tissues, including heart and brain. Within the brain, relaxin is expressed in the olfactory and limbic systems, the cortex and the hypothalamic arcuate nucleus (ARC). Its cognate receptor, relaxin family peptide receptor 1 (RXFP1), is also widely expressed in the brain, including the hypothalamic ARC and paraventricular nucleus (PVN), areas important in appetite regulation. The aim of this study was to investigate whether relaxin influences food intake through central hypothalamic circuits. METHODS: The human form of relaxin, human relaxin-2 (H2) was administered centrally and peripherally to male Wistar rats and food intake measured. Behaviour was also assessed. RESULTS: Intracerebroventricular (ICV) administration of H2 significantly decreased 1-h food intake in the early dark phase [2.95 ± 0.45 g (saline) vs. 0.95 ± 0.18 g (180 pmol H2), p < 0.001]. ICV administration of H2 decreased feeding behaviour and increased grooming and headdown behaviour. Intraparaventricular injections of H2 significantly decreased 1-h food intake in the early dark phase [3.13 ± 0.35 g (saline) vs. 1.35 ± 0.33 g (18 pmol H2), p < 0.01, 1.61 ± 0.31 g (180 pmol H2), p < 0.05 and 1.23 ± 0.32 g (540 pmol H2), p < 0.001]. Intraperitoneal (IP) administration of H2 significantly decreased 1-h food intake in the early dark phase [4.63 ± 0.46 g (vehicle) vs. 3.08 ± 0.15 g (66 nmol H2), p < 0.01, 3.00 ± 0.17 g (200 nmol H2), p < 0.01 and 2.26 ± 0.36 g (660 nmol H2), p < 0.001]. CONCLUSIONS: Central and peripheral administration of H2 reduces the food intake in rats. This effect may be mediated via the PVN and/or other brain regions.

Cerebellin1 is a novel orexigenic peptide.

AIM: Cerebellin1 (Cbln1) is highly expressed in the hypothalamus, a region of the brain involved in appetite regulation. However, the effects of Cbn1 on food intake are not known. The present study aimed to investigate the effect of Cbln1 on appetite regulation in rats. METHODS: We determined the effect of (i) intracerebroventricular (ICV) injection of Cbln1 on food intake, behaviour and plasma pituitary hormone levels in male Wistar rats; (ii) Cbln1 on the release of hypothalamic neuropeptides known to modulate food intake from hypothalamic explants and (iii) fasting on hypothalamic Cbln1 mRNA expression. RESULTS: (i) ICV administration of Cbln1 significantly increased food intake in rats and caused no adverse behaviours. ICV administration of Cbln1 significantly reduced plasma thyroid stimulating hormone (TSH) levels 10 min postinjection in rats. (ii) Cbln1 significantly increased the release of neuropeptide Y (NPY) from hypothalamic explants. (iii) Cbln1 mRNA expression levels were increased in the ventromedial nucleus of the hypothalamus in fasted rats. CONCLUSIONS: These data suggest that Cbln1 is a novel orexigenic peptide, which may mediate its effects via hypothalamic NPY.

Energetics and kinematics of walking in the barnacle goose (Branta leucopsis).

Barnacle geese were walked on a treadmill at speeds ranging from 0.25 to 1.25 ms(-1), which was their highest sustainable speed. No evidence for a gait change was found. The gait of a barnacle goose appears to conform to the classical pendulum mechanics based model of walking, with the kinetic energy of forward motion (horizontal kinetic energy, E(kh)) out-of-phase with the sum of the gravitational potential (E(p)), and vertical kinetic (E(kv)) energies of the centre of mass at all speeds. Why barnacle geese are unable to aerial run when other 'waddling' species do show an aerial phase (e.g., mallard ducks) is unclear. Presumably, however, it is likely to relate to the amount of lateral kinetic energy generated, which is a feature of 'waddling'. We predict that lateral kinetic energy generated by barnacle geese and other waddling species that cannot aerial run, is higher than in those that can. Due to competing selection pressures for swimming and flight, barnacle geese are mechanically and energetically inefficient walkers relative to more specialist cursorial birds. Their upper walking speed, however, appears to be limited by morphology (via kinematics) and not metabolic capacity (energetics).

Fifty ways to improve presentations in urology. / Cincuenta consejos para mejorar las presentaciones en urología.

BACKGROUND: Our profession permanently demands intercommunication of medical knowledge among colleagues; either in small environments such as hospitals or at larger ones such as congresses or academic courses. New technologies such as PowerPoint® are not developed enough to provide good presentations, and its employment does not always grant effective results. OBJECTIVE: In order to improve our academic presentations, we present several tools that may help us avoid the most common mistakes. EVIDENCE ACQUISITION: Literature search in PubMed and Google Scholar. We have divided the analysis into 3 sections: structure of the presentation, slide design, presentation to the audience. Each section includes a list of 50 short tips. RESULTS: Fifty tips following the study objectives. CONCLUSIONS: The scientific evidence that supports the information on how to improve presentations is mostly based on expert opinions. However, almost every work agrees that presentations must use simple structures which does not make them less scientific; their content must be developed for a specific audience, and it must be the speaker, not the slides, who captures the audience attention. Making a simple and didactic presentation of complex content supported by multimedia tools is one of the speaker's highest intellectual challenges of these days.

Towards correlative imaging of plant cortical microtubule arrays: combining ultrastructure with real-time microtubule dynamics.

There are a variety of microscope technologies available to image plant cortical microtubule arrays. These can be applied specifically to investigate direct questions relating to array function, ultrastructure or dynamics. Immunocytochemistry combined with confocal laser scanning microscopy provides low resolution "snapshots" of cortical microtubule arrays at the time of fixation whereas live cell imaging of fluorescent fusion proteins highlights the dynamic characteristics of the arrays. High-resolution scanning electron microscopy provides surface detail about the individual microtubules that form cortical microtubule arrays and can also resolve cellulose microfibrils that form the innermost layer of the cell wall. Transmission electron microscopy of the arrays in cross section can be used to examine links between microtubules and the plasma membrane and, combined with electron tomography, has the potential to provide a complete picture of how individual microtubules are spatially organized within the cortical cytoplasm. Combining these high-resolution imaging techniques with the expression of fluorescent cytoskeletal fusion proteins in live cells using correlative microscopy procedures will usher in an radical change in our understanding of the molecular dynamics that underpin the organization and function of the cytoskeleton.