Maternal Larp6 controls oocyte development, chorion formation and elevation.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.

A Machine Learning-Based Image Segmentation Method to Quantify In Vitro Osteoclast Culture Endpoints.

Quantification of in vitro osteoclast cultures (e.g. cell number) often relies on manual counting methods. These approaches are labour intensive, time consuming and result in substantial inter- and intra-user variability. This study aimed to develop and validate an automated workflow to robustly quantify in vitro osteoclast cultures. Using ilastik, a machine learning-based image analysis software, images of tartrate resistant acid phosphatase-stained mouse osteoclasts cultured on dentine discs were used to train the ilastik-based algorithm. Assessment of algorithm training showed that osteoclast numbers strongly correlated between manual- and automatically quantified values (r = 0.87). Osteoclasts were consistently faithfully segmented by the model when visually compared to the original reflective light images. The ability of this method to detect changes in osteoclast number in response to different treatments was validated using zoledronate, ticagrelor, and co-culture with MCF7 breast cancer cells. Manual and automated counting methods detected a 70% reduction (p < 0.05) in osteoclast number, when cultured with 10 nM zoledronate and a dose-dependent decrease with 1-10 µM ticagrelor (p < 0.05). Co-culture with MCF7 cells increased osteoclast number by ≥ 50% irrespective of quantification method. Overall, an automated image segmentation and analysis workflow, which consistently and sensitively identified in vitro osteoclasts, was developed. Advantages of this workflow are (1) significantly reduction in user variability of endpoint measurements (93%) and analysis time (80%); (2) detection of osteoclasts cultured on different substrates from different species; and (3) easy to use and freely available to use along with tutorial resources.

Restraint upon Embryonic Metatarsal Ex Vivo Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway.

Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.

BACKGROUND: Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. RESULTS: Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. CONCLUSIONS: Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

ERK2 but not ERK1 mediates HGF-induced motility in non-small cell lung carcinoma cell lines.

Aberrant signalling of receptor tyrosine kinases (RTKs), such as c-Met, the receptor for hepatocyte growth factor (HGF), has been implicated in the oncogenesis of various tumours including non-small cell lung carcinoma (NSCLC). Through its pro-migratory properties, c-Met has been implicated specifically in the process of tumour metastasis, demanding a better understanding of the underlying signalling pathways. Various players downstream of c-Met have been well characterised, including the extracellular-signal-regulated kinases (ERKs) 1 and 2. In a small interfering RNA (siRNA)-based high-throughput wound healing screen performed in A549 lung carcinoma cells, we identified ERK2 but not ERK1 as a strong mediator of HGF-induced motility. This finding was confirmed in several NSCLC cell lines as well as in HeLa cells. One known substrate for ERK kinases in cell migration, the focal adhesion protein paxillin, was also one of the hits identified in the screen. We demonstrate that HGF stimulation results in a time-dependent phosphorylation of paxillin on serine 126, a process that can be blocked by inhibition of the ERK1/2 upstream kinase mitogen-activated protein kinase/ERK kinase 1 (MEK1) or inhibition of glycogen synthase kinase 3 (GSK3). Further, we show that paxillin turnover at focal adhesions is increased upon stimulation by HGF, an effect that is dependent on serine residues 126 (GSK3 site) and 130 (ERK site) within paxillin. In line with the isoform-specific requirement of ERK2 for HGF-mediated migration in lung tumour cell models, ERK2 but not ERK1 is shown to be responsible for paxillin serine 126 phosphorylation and its increased turnover at focal adhesions.

A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 µl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 µl/cm(2) in 24-well (10 µl) or 96-well (2 µl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.

Performance Kinetics During Repeated Sprints is Influenced by Knowledge of Task Endpoint and Associated Peripheral Fatigue.

The regulation of exercise intensity allows an athlete to perform an exercise in the fastest possible time while avoiding debilitating neuromuscular fatigue development. This phenomenon is less studied during intermittent activities. To investigate anticipatory and real-time regulation of motor output and neuromuscular fatigue during repeated-sprint exercise, twelve males randomly performed one (S1), two (S2), four (S4) and six (S6) sets of five 5-s cycling sprints. Mechanical work and electromyographic activity were assessed during sprints. Potentiated quadriceps twitch force (ΔQtw,pot) and central activation ratio (QCAR) were quantified from response to supra-maximal magnetic femoral nerve stimulation pre-vs post-exercise. Compared with S1, mechanical work developed in the first sprint and in the entire first set was reduced in S6 (-7.8% and -5.1%, respectively, P < 0.05). Work developed in the last set was similar in S4 and S6 (P = 0.82). Similar results were observed for EMG activity. The QCAR was also more reduced in S4 (-5.8%, P < 0.05) and S6 (-8.3%, P < 0.05) than in S1. However, ΔQtw,pot was not significantly different across all trials (-33.1% to -41.9%, P = 0.46). Perceived exhaustion increased across sprints to reach a maximal and similar level in S2, S4 and S6 (all 19.2, P < 0.01 vs S1). These results suggest that the regulation of performance, exerted at the beginning and continuously during repeated sprints, is based on the task endpoint, presumably to avoid excessive peripheral muscle and associated conscious overwhelming sensations.

Goal Orientation and the Presence of Competitors Influence Cycling Performance.

Introduction: The aim of this study was to investigate time-trial (TT) performance in the presence of one competitor and in a group with competitors of various abilities. Methods: In a randomized order, 24 participants performed a 5-km cycling TT individually (IND), with one similarly matched participant (1v1), and in a group of four participants (GRP). For the GRP session, two pairs of matched participants from the 1v1 session were used. Pairs were selected so that TT duration was considered either inferior (INF) or superior (SUP) compared to the other pair of participants. Results: Overall, TT duration (P = 0.86, ηp2 < 0.01) was not different between conditions, while heart rate (HR) was significantly greater in GRP compared to IND (P < 0.01, ηp2 = 0.16). For INF, a large effect size for both mean power (P = 0.07, ηp2 = 0.15) and HR (P = 0.05, ηp2 = 0.16), indicates greatest effort in GRP. Pacing behavior was affected by competition but similar in 1v1 and GRP for SUP, while large effect sizes indicate an increased power output in the initial 750-m for INF in GRP. Additionally, for INF, there was a significant correlation with ego orientation for an increase in TT duration between the GRP session and both the IND (r = 0.43, P = 0.04) and 1v1 (r = 0.54, P = 0.01) sessions. Conclusion: For INF participants, intensity was increased when competing in GRP. Yet, the presence of the SUP competitors resulted in lesser performance improvements for ego oriented INF participants. These findings demonstrate that consideration should be given to the ability of competitors in a group setting to provide adequate motivation.

Familiarization Protocol Influences Reproducibility of 20-km Cycling Time-Trial Performance in Novice Participants.

Introduction: Exercise performance is reproducible in experienced athletes; however, less trained participants exhibit greater variability in performance and pacing. To reduce variability, it is common practice to complete a familiarization prior to experimental testing. However, there are no clear guidelines for familiarizing novice participants to a cycling time-trial (TT), and research findings from novice populations may still be influenced by learning effects. Accordingly, the aims of this study were to establish the variability between TTs after administering differing familiarization protocols (duration or type) and to establish the number of familiarization trials required to limit variability over multiple trials. Methods: Thirty recreationally active participants, with no prior experience of a TT, performed a 20-km cycling TT on five separate occasions, after completing either a full (FF, 20-km TT, n = 10), a half (HF, 10-km TT, n = 10) or an equipment familiarization (EF, 5-min cycling, n = 10). Results: Variability of TT duration across five TTs was the lowest after completing FF (P = 0.69, η p2 = 0.05) compared to HF (P = 0.08, η p2 = 0.26) and EF (P = 0.07, η p2 = 0.21). In the FF group after TT2, the effect size for changes in TT duration was small (d < 0.49). There were large differences between later TTs in HF (d = 1.02, TT3-TT4) and EF (d = 1.12, TT4-TT5). The variability in mean power output profiles between trials was lowest within FF, with a similar pacing profile reproduced between TT3-TT5. Discussion: Familiarization of the exercise protocol influenced reproducibility of pacing and performance over multiple, maximal TTs, with best results obtained after a full experience of the exercise compared to HF and EF. The difference of TT1 to later TTs indicates that one familiarization is not adequate in reducing the variability of performance for novice participants. After the FF and an additional TT, performance changes between TTs were small, however, a reproducible pacing profile was not developed until after the FF and two additional TTs. These findings indicate that a minimum of three full familiarizations are necessary for novice participants to limit systematic error before experimental testing.

No Influence of Transcutaneous Electrical Nerve Stimulation on Exercise-Induced Pain and 5-Km Cycling Time-Trial Performance.

Introduction: Afferent information from exercising muscle contributes to the sensation of exercise-induced muscle pain. Transcutaneous electrical nerve stimulation (TENS) delivers low-voltage electrical currents to the skin, inhibiting nociceptive afferent information. The use of TENS in reducing perceptions of exercise-induced pain has not yet been fully explored. This study aimed to investigate the effect of TENS on exercise-induced muscle pain, pacing strategy, and performance during a 5-km cycling time trial (TT). Methods: On three separate occasions, in a single-blind, randomized, and cross-over design, 13 recreationally active participants underwent a 30-min TENS protocol, before performing a 5-km cycling TT. TENS was applied to the quadriceps prior to exercise under the following conditions; control (CONT), placebo with sham TENS application (PLAC), and an experimental condition with TENS application (TENS). Quadriceps fatigue was assessed with magnetic femoral nerve stimulation assessing changes in potentiated quadriceps twitch force at baseline, pre and post exercise. Subjective scores of exertion, affect and pain were taken every 1-km. Results: During TTs, application of TENS did not influence pain perceptions (P = 0.68, [Formula: see text] = 0.03). There was no significant change in mean power (P = 0.16, [Formula: see text] = 0.16) or TT duration (P = 0.17, [Formula: see text] = 0.14), although effect sizes were large for these two variables. Changes in power output were not significant but showed moderate effect sizes at 500-m ([Formula: see text] = 0.10) and 750-m ([Formula: see text] = 0.10). Muscle recruitment as inferred by electromyography data was not significant, but showed large effect sizes at 250-m ([Formula: see text] = 0.16), 500-m ([Formula: see text] = 0.15), and 750-m ([Formula: see text] = 0.14). This indicates a possible effect for TENS influencing performance up to 1-km. Discussion: These findings do not support the use of TENS to improve 5-km TT performance.

Myosin VI and Associated Proteins Are Expressed in Human Macrophages but Do Not Play a Role in Foam Cell Formation in THP-1 Cells.

Myosin VI (Myo6) functions in endocytosis in conjunction with binding partners including adaptor protein (AP)-2, disabled 2 (Dab2), and GAIP interacting protein C terminus 1 (GIPC1). This study aimed to investigate the expression and function of Myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation. Expression of Myo6, AP-2 ( α 2 subunit), and Dab2 in THP-1 macrophages and primary human monocyte-derived macrophages was demonstrated at the mRNA and protein level, but GIPC1 was only detected at the mRNA level. Immunofluorescence showed that Myo6 was distributed similarly to F-actin in both macrophage types. AP-2 α 2 was found to have a similar subcellular distribution to Myo6 and Dab2 in THP-1 cells. Myo6 was located within membrane ruffles and protrusions of the plasma membrane. These results suggest that in macrophages Myo6 is required for several functions including cell adhesion, cell progression, and macropinocytosis. Low-density lipoprotein (LDL) and oxidised LDL (oxLDL) decreased Myo6 and GIPC1 mRNA expression in THP-1 cells, but uptake of the fluorescence-labelled lipoproteins was unaffected by knockdown of the expression of Myo6 or associated proteins with siRNA. Our findings, therefore, do not support the idea that Myo6 plays a major role in foam cell formation.

Enterococcus faecalis subverts and invades the host urothelium in patients with chronic urinary tract infection.

Bacterial urinary tract infections (UTI) are a major growing concern worldwide. Uropathogenic Escherichia coli has been shown to invade the urothelium during acute UTI in mice and humans, forming intracellular reservoirs that can evade antibiotics and the immune response, allowing recurrence at a later date. Other bacterial species, such as Staphylococcus saprophyticus, Klebsiella pneumonia and Salmonella enterica have also been shown to be invasive in acute UTI. However, the role of intracellular infection in chronic UTI causing more subtle lower urinary tract symptoms (LUTS), a particular problem in the elderly population, is poorly understood. Moreover, the species of bacteria involved remains largely unknown. A previous study of a large cohort of non-acute LUTS patients found that Enterococcus faecalis was frequently found in urine specimens. E. faecalis accounts for a significant proportion of chronic bladder infections worldwide, although the invasive lifestyle of this uropathogen has yet to be reported. Here, we wanted to explore this question in more detail. We harvested urothelial cells shed in response to inflammation and, using advanced imaging techniques, inspected them for signs of bacterial pathology and invasion. We found strong evidence of intracellular E. faecalis harboured within urothelial cells shed from the bladder of LUTS patients. Furthermore, using a culture model system, these patient-isolated strains of E. faecalis were able to invade a transitional carcinoma cell line. In contrast, we found no evidence of cellular invasion by E. coli in the patient cells or the culture model system. Our data show that E. faecalis is highly competent to invade in this context; therefore, these results have implications for both the diagnosis and treatment of chronic LUTS.

Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair.

Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of CD172a (pan Mφ), CD14(high)CD206(low) (pro-inflammatory M1Mφ), and CD206(high) (anti-inflammatory M2Mφ) to assess potential polarised phenotypes. In addition, the Lipoxin A(4) receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mφ and FPR2/ALX. In contrast, M1Mφ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mφ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mφ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A(4) (LXA(4)) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E(2). Stimulation with either mediator induced LXA(4) release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mφ.

Protein kinase C intervention: the state of play.

Intervention in protein kinase C (PKC) has a chequered history, partly because of the poor selectivity of many inhibitors and partly a reflection of the sometimes antagonistic action of related PKC isoforms. Recent advances in targeting PKC isoforms have come from structural work on isolated kinase domains that have provided opportunities to drive selectivity through structure-based avenues. The promise of isoform selective inhibitors and the rationale for their development are discussed in the broader context of the PKC inhibitor arsenal.

The localization, trafficking and retrograde transport of BDNF bound to p75NTR in sympathetic neurons.

BDNF, through p75NTR, promotes apoptosis and inhibits axonal growth of sympathetic neurons, antagonizing the pro-survival and axon growth-promoting actions of NGF through TrkA. While the trafficking of the TrkA:NGF complex is well characterized, little is known about p75NTR:BDNF trafficking in these neurons. Here we show that BDNF binds to and appears inside sympathetic neurons relatively slowly, although the temperature-sensitive internalization step itself is rapid. P75NTR internalization is partially sensitive to disruption of clathrin- or raft-mediated internalization, while that of TrkA is entirely clathrin-mediated. P75NTR, but not Trk, associates with neurotrophins in lipid rafts and coimmunoprecipitates with the truncated beta-caveolin-1 isoform. Finally, we directly visualize the retrograde transport of p75NTR ligands to cell bodies, which is insensitive to inhibitors of Trk retrograde transport, suggesting mechanistic differences. We postulate that beta-caveolin-1-containing lipid rafts and possibly intracellular endosomes might be compartments to which p75NTR:BDNF complexes are trafficked separately from Trk.

Neurotrophin-4, alone or heterodimerized with brain-derived neurotrophic factor, is sorted to the constitutive secretory pathway.

Nerve growth factor and neurotrophin-3 (NT-3) are processed within the constitutive secretory pathway of neurons and neuroendocrine cells and are released continuously in an activity-independent fashion. In contrast, brain-derived neurotrophic factor (BDNF) is processed in the regulated secretory pathway, stored in vesicles, and released in response to neuronal activity, consistent with its role in modulating synaptic plasticity. In this study, we used vaccinia virus infection and transfection methods to monitor the processing and sorting of neurotrophin-4 (NT-4) in AtT-20 cells, which have been used as a model for the sorting of secretory proteins in neurons. Our data show that NT-4 is processed in the constitutive secretory pathway. The molecule is diffusely distributed within the cells and released, soon after being synthesized, in a manner that is not affected by cell depolarization. We further show that NT-4 and BDNF, when co-expressed, can form heterodimers that are constitutively released. In contrast, heterodimers of NT-3 and BDNF have been shown to be released through the regulated secretory pathway. Thus, NT-4, alone or when co-expressed with BDNF, is processed within and secreted by the constitutive secretory pathway.