Proximal Compartment Research Articles

Distinct Neural Drives along the Semitendinosus Muscle.

Conflicting results have been reported on the functional role of the proximal and distal compartments of the semitendinosus (ST) muscle. This study compared the discharge characteristics of motor units (MUs) in the two compartments at three knee-joint angles (0°: long length, 45°: intermediate length, and 90°: short length). Twenty men (21.4 ± 2.3 years) performed steady isometric contractions with the knee flexors at four target forces: 10%, 20%, 40%, and 60% of maximum voluntary contraction (MVC). High-density electromyography (EMG) signals were recorded to examine the MU discharge characteristics in the two compartments. Measurements included recruitment threshold (RT), mean discharge rate (MDR), coefficient of variation for interspike interval (CoV for ISI), and the standard deviation of filtered cumulative spike train (SD of fCST). Additionally, the within- and between-compartment association of the neural drive was calculated. Analysis of variance indicated that maximal force, absolute EMG amplitude during the MVCs, and force steadiness (CoV for force) were greater at the longest muscle length than the other two lengths (P < 0.05). Linear mixed models showed that both RT and CoV for ISI were similar between compartments (P > 0.05) at each of the three knee-joint angles. However, MDR and the variability in neural drive were greater for the proximal than the distal compartment (P < 0.05). The between-compartment association in neural drive (fCST) was relatively low. There were distinct differences in motor unit discharge characteristics between the proximal and distal compartments of ST across its operating range of muscle lengths and each compartment received a relatively distinct neural drive. These findings emphasize the importance of recognizing differences in neural control of the ST compartments to guide related interventions and inform rehabilitation strategies.

Human tripartite cortical network model for temporal assessment of alpha-synuclein aggregation and propagation in Parkinson’s Disease

Previous studies have shown that aggregated alpha-synuclein (α-s) protein, a key pathological marker of Parkinson’s disease (PD), can propagate between cells, thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models, including rodent and human cell cultures. In this study, our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here, we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore, aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells, such as in presynaptic proteins, mitochondrial motility, calcium oscillations and neuronal activity. The model enabled an assessment of the early, middle, and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates, further investigation is required to elucidate the underlying mechanisms. Taken together, this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Exploring Kainic Acid-Induced Alterations in Circular Tripartite Networks with Advanced Analysis Tools.

Brain activity implies the orchestrated functioning of interconnected brain regions. Typical in vitro models aim to mimic the brain using single human pluripotent stem cell-derived neuronal networks. However, the field is constantly evolving to model brain functions more accurately through the use of new paradigms, e.g., brain-on-a-chip models with compartmentalized structures and integrated sensors. These methods create novel data requiring more complex analysis approaches. The previously introduced circular tripartite network concept models the connectivity between spatially diverse neuronal structures. The model consists of a microfluidic device allowing axonal connectivity between separated neuronal networks with an embedded microelectrode array to record both local and global electrophysiological activity patterns in the closed circuitry. The existing tools are suboptimal for the analysis of the data produced with this model. Here, we introduce advanced tools for synchronization and functional connectivity assessment. We used our custom-designed analysis to assess the interrelations between the kainic acid (KA)-exposed proximal compartment and its nonexposed distal neighbors before and after KA. Novel multilevel circuitry bursting patterns were detected and analyzed in parallel with the inter- and intracompartmental functional connectivity. The effect of KA on the proximal compartment was captured, and the spread of this effect to the nonexposed distal compartments was revealed. KA induced divergent changes in bursting behaviors, which may be explained by distinct baseline activity and varied intra- and intercompartmental connectivity strengths. The circular tripartite network concept combined with our developed analysis advances importantly both face and construct validity in modeling human epilepsy in vitro.

Reelin differentially shapes dendrite morphology of medial entorhinal cortical ocean and island cells.

The function of medial entorhinal cortex layer II (MECII) excitatory neurons has been recently explored. MECII dysfunction underlies deficits in spatial navigation and working memory. MECII neurons comprise two major excitatory neuronal populations, pyramidal island and stellate ocean cells, in addition to the inhibitory interneurons. Ocean cells express reelin and surround clusters of island cells that lack reelin expression. The influence of reelin expression by ocean cells and interneurons on their own morphological differentiation and that of MECII island cells has remained unknown. To address this, we used a conditional reelin knockout (RelncKO) mouse to induce reelin deficiency postnatally in vitro and in vivo. Reelin deficiency caused dendritic hypertrophy of ocean cells, interneurons and only proximal dendritic compartments of island cells. Ca2+ recording showed that both cell types exhibited an elevation of calcium frequencies in RelncKO, indicating that the hypertrophic effect is related to excessive Ca2+ signalling. Moreover, pharmacological receptor blockade in RelncKO mouse revealed malfunctioning of GABAB, NMDA and AMPA receptors. Collectively, this study emphasizes the significance of reelin in neuronal growth, and its absence results in dendrite hypertrophy of MECII neurons.

Structure dependent fermentation kinetics of dietary carrot rhamnogalacturonan-I in an in vitro gut model

Plant derived dietary polysaccharides are important for gut health and have the potential to modulate the gut microbial community. Dietary rhamnogalacturonan-I obtained by enzymatic treatment of carrot pomace has shown prebiotic properties. In the present study, fermentability of carrot rhamnogalacturonan-I (cRG-I) by faecal microbiota of four donors was studied in an adapted M-SHIME® intestinal model. Despite its complex structure, cRG-I was degraded rapidly in the proximal colon compartment and fermentation became quicker and more complete during three weeks of repeated supplementation. Tracking the change in the molecular weight distribution pattern of cRG-I during the supplementation showed two main donor-dependent gut microbial fermentation strategies designated as either the general or preferential pathway. In the general fermentation pathway, different cRG-I structures were hydrolysed concomitantly, while in the preferential pathway discrete structures were sequentially fermented in a selective manner. Especially arabinan sidechains were utilized before the RG-I backbone, which correlated with an increase in Bifidobacterium longum absolute abundance over the three weeks period. MALDI-TOF MS confirmed that arabinan-, galactan- and arabinogalactan-sidechains were first to be released and degraded. Donor specific production of all SCFA increased over time with a general trend of higher levels of acetate and propionate than butyrate. Strikingly, although the host's baseline gut microbiota composition led to distinct cRG-I hydrolysis routes, the final RG-I consumption was almost complete for both routes, leading to similar metabolic profiles at the end of the three weeks treatment period.

Somatodendritic organization of pacemaker activity in midbrain dopamine neurons.

The slow and regular pacemaking activity of midbrain dopamine (DA) neurons requires proper spatial organization of the excitable elements between the soma and dendritic compartments, but the somatodendritic organization is not clear. Here, we show that the dynamic interaction between the soma and multiple proximal dendritic compartments (PDCs) generates the slow pacemaking activity in DA neurons. In multipolar DA neurons, spontaneous action potentials (sAPs) consistently originate from the axon-bearing dendrite. However, when the axon initial segment was disabled, sAPs emerge randomly from various primary PDCs, indicating that multiple PDCs drive pacemaking. Ca2+ measurements and local stimulation/perturbation experiments suggest that the soma serves as a stably-oscillating inertial compartment, while multiple PDCs exhibit stochastic fluctuations and high excitability. Despite the stochastic and excitable nature of PDCs, their activities are balanced by the large centrally-connected inertial soma, resulting in the slow synchronized pacemaking rhythm. Furthermore, our electrophysiological experiments indicate that the soma and PDCs, with distinct characteristics, play different roles in glutamate- induced burst-pause firing patterns. Excitable PDCs mediate excitatory burst responses to glutamate, while the large inertial soma determines inhibitory pause responses to glutamate. Therefore, we could conclude that this somatodendritic organization serves as a common foundation for both pacemaker activity and evoked firing patterns in midbrain DA neurons.

Advanced Immunolabeling Method for Optical Volumetric Imaging Reveals Dystrophic Neurites of Dopaminergic Neurons in Alzheimer’s Disease Mouse Brain

Optical brain clearing combined with immunolabeling is valuable for analyzing molecular tissue structures, including complex synaptic connectivity. However, the presence of aberrant lipid deposition due to aging and brain disorders poses a challenge for achieving antibody penetration throughout the entire brain volume. Herein, we present an efficient brain-wide immunolabeling method, the immuno-active clearing technique (iACT). The treatment of brain tissues with a zwitterionic detergent, specifically SB3-12, significantly enhanced tissue permeability by effectively mitigating lipid barriers. Notably, Quadrol treatment further refines the methodology by effectively eliminating residual detergents from cleared brain tissues, subsequently amplifying volumetric fluorescence signals. Employing iACT, we uncover disrupted axonal projections within the mesolimbic dopaminergic (DA) circuits in 5xFAD mice. Subsequent characterization of DA neural circuits in 5xFAD mice revealed proximal axonal swelling and misrouting of distal axonal compartments in proximity to amyloid-beta plaques. Importantly, these structural anomalies in DA axons correlate with a marked reduction in DA release within the nucleus accumbens. Collectively, our findings highlight the efficacy of optical volumetric imaging with iACT in resolving intricate structural alterations in deep brain neural circuits. Furthermore, we unveil the compromised integrity of DA pathways, contributing to the underlying neuropathology of Alzheimer’s disease. The iACT technique thus holds significant promise as a valuable asset for advancing our understanding of complex neurodegenerative disorders and may pave the way for targeted therapeutic interventions.Graphical The axonal projection of DA neurons in the septum and the NAc showed dystrophic phenotypes such as growth cone-like enlargement of the axonal terminus and aggregated neurites. Brain-wide imaging of structural defects in the neural circuits was facilitated with brain clearing and antibody penetration assisted with SB3-12 and Quadrol pre-treatment. The whole volumetric imaging process could be completed in a week with the robust iACT method. Created with https://www.biorender.com/.

Regional and disease-specific glycosaminoglycan composition and function in decellularized human lung extracellular matrix.

Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor β to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease.

Nicotinamide N-Methyl Transferase as a Predictive Marker of Tubular Fibrosis in CKD.

Chronic kidney disease (CKD) progression is complex. There are not standardized methods for predicting the prognosis of CKD. Nicotinamide N-methyltransferase (NNMT) has been shown to be associated with renal fibrosis. This study aimed to validate NNMT as a prognostic biomarker of progressive CKD. We explored the relationship between NNMT expression and CKD-related outcome variables using the NephroseqV5 and GEO databases. Additionally, a validation set of 37 CKD patients was enrolled to measure the correlation between NNMT expression levels and CKD outcomes. Furthermore, single-cell RNA sequencing data and the Human Protein Atlas were reanalyzed to investigate the expression specificity of NNMT in the kidney. Finally, to detect the status of NNMT expression with tubular fibrosis in vivo, we constructed a unilateral ureteral obstruction (UUO) mouse treated with an NNMT inhibitor. Analyzing the datasets showed that NNMT was expressed mainly in proximal tubule compartments. And patients with high NNMT expression levels had a significantly lower overall survival rate compared to those with low NNMT expression levels (P = 0.013). NNMT was independent of prognosis factors in the multivariate Cox regression model, and the AUCs for CKD progression at 1, 3, and 5 years were 0.849, 0.775, and 0.877, respectively. Pathway enrichment analysis indicated that NNMT regulates the biological processes of tubulointerstitial fibrosis (TIF). In the validation group, NNMT levels were significantly higher in the CKD group combined with interstitial fibrosis. In vivo, NNMT was a high expression in the UUO group, peaking at postoperative day 21. Treatment with an NNMT inhibitor improved renal tubular interstitial fibrosis, and expression levels of FN, α-SMA, VIM, and TGF-β1 were decreased compared with UUO (P < 0.05). NNMT was expressed mainly in tubular renal compartments, and associated with CKD prognosis. It holds potential as a diagnostic biomarker for tubular fibrosis in CKD.

Novel In Vitro Intestinal Microbiome Model to Study Lipidomic and Metabolomic Adaptations Due to Exposure to Glyphosate, Perfluorooctanoic Acid, and Docusate Sodium.

Cell and animal models have been used to provide insights with regard to physiological changes in intestinal flora due to exposure to drugs and environmental contaminants. Here, a novel in vitro model known as simulator of the human intestinal microbial ecosystem (SHIME) was used to assess the effects of three chemicals of emerging concern, namely glyphosate, perfluorooctanoic acid (PFOA), and docusate sodium (dioctyl sulfosuccinate, DOSS), on the lipidomic and metabolomic profiles of the gut microenvironment in both the proximal and distal colonic compartments. Nontargeted analyses by ultra-high performance liquid chromatography-tandem mass spectrometry and gas chromatography-electron ionization-mass spectrometry revealed minor differences in the lipidomic and metabolomic signatures of the proximal and distal colon following treatment with either glyphosate or PFOA at acceptable human daily intake levels or average daily exposures. However, global dysregulation of lipids and metabolites was observed due to DOSS treatment at conventional prescription doses when indicated as a stool softener. Our findings suggest that the current guidelines for glyphosate and PFOA exposure may be adequate at the level of the lower gut microbiome in healthy adults, but the probable yet uncharacterized off-target effects, safety, and efficacy of long-term DOSS treatment warrants further investigation. Indeed, we highlight the SHIME system as a novel in vitro approach which can be used as a screening tool to assess the impact of drugs and/or chemicals on the gut microbiome, while implementing state-of-the-art and data-driven mass spectrometric workflows to identify toxic lipidomic and metabolomic signatures.

Modelling renal defects in Bardet-Biedl syndrome patients using human iPS cells.

Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney.

Morphology of proximal and distal human semitendinosus compartments and the effects of distal tendon harvesting for anterior cruciate ligament reconstruction.

The human semitendinosus muscle is characterized by a tendinous inscription separating proximal and distal neuromuscular compartments. As each compartment is innervated by separate nerve branches, potential exists for independent operation and control of compartments. However, the morphology and function of each compartment have not been thoroughly examined in an adult human population. Further, the distal semitendinosus tendon is typically harvested for use in anterior cruciate ligament reconstruction surgery, which induces long-term morphological changes to the semitendinosus muscle-tendon unit. It remains unknown if muscle morphological alterations following anterior cruciate ligament reconstruction are uniform between proximal and distal semitendinosus compartments. Here, we performed magnetic resonance imaging on 10 individuals who had undergone anterior cruciate ligament reconstruction involving an ipsilateral distal semitendinosus tendon graft 14 ± 4 months prior, extracting morphological parameters of the whole semitendinosus muscle and each individual compartment from both the (non-injured) contralateral and surgical legs. In the contralateral leg, volume and length of the proximal compartment were smaller than the distal compartment. No between-compartment differences in volume or length were found for anterior cruciate ligament reconstructed legs, likely due to greater shortening of the distal compared to the proximal compartment after anterior cruciate ligament reconstruction. The maximal anatomical cross-sectional area of both compartments was substantially smaller on the anterior cruciate ligament reconstructed leg but did not differ between compartments on either leg. The absolute and relative between-leg differences in proximal compartment morphology on the anterior cruciate ligament reconstructed leg were strongly correlated with the corresponding between-leg differences in distal compartment morphological parameters. Specifically, greater between-leg morphological differences in one compartment were highly correlated with large between-leg differences in the other compartment, and vice versa for smaller differences. These relationships indicate that despite the heterogeneity in compartment length and volume, compartment atrophy is not independent or random. Further, the tendinous inscription endpoints were generally positioned at the same proximodistal level as the compartment maximal anatomical cross-sectional areas, providing a wide area over which the tendinous inscription could mechanically interact with compartments. Overall, results suggest the two human semitendinosus compartments are not mechanically independent.

First extensor compartment morphology and clinical significance: a cadaver series study.

The first extensor compartment of the wrist is a distinctly variable anatomical area. Anatomical variations in this region contribute to the pathophysiology and treatment failure of de Quervain's disease, which is a kind of tenosynovitis that develops in the first extensor compartment of the wrist. We aim to describe the first extensor compartment morphology, to evaluate the septum frequency, location of the septum, and the number of tendons of abductor pollicis longus (APL) and extensor pollicis brevis muscles (EPB). First extensor compartment of 87 wrists of 45 cadavers were dissected. The presence or absence of septum and number of tendon slips of APL and EPB revealed. The proximal and distal widths of the compartments were measured. Septums were detected in 60.9% (n=53) of the wrists. Incomplete (distal) and complete (proximal) septa were present in 35.6% (n=31) and 25.3% (n=22) of the cases. Only 26.4% of the wrists had a single slip of APL tendon. The Remaining had multiple slips. The median inner width of the proximal and distal compartments in all wrists were calculated as in the order of 9.11±1.14 mm and 8.55±1.12 mm. We believe that understanding the anatomy of the first extensor compartment in the Turkish population would be helpful to surgeons, radiologists, and physiotherapists to diagnose and manage de Quervain's disease.

Proximal dendritic localization of NALCN channels underlies tonic and burst firing in nigral dopaminergic neurons.

In multipolar nigral dopamine (DA) neurons, the highly excitable proximal dendritic compartments (PDCs) and two Na+ -permeable leak channels, TRPC3 and NALCN, play a key role in pacemaking. However, the causal link between them is unknown. Here we report that the proximal dendritic localization of NALCN underlies pacemaking and burst firing in DA neurons. Our morphological analysis of nigral DA neurons reveals that TRPC3 is ubiquitously expressed in the whole somatodendritic compartment, but NALCN is localized within the PDCs. Blocking either TRPC3 or NALCN channels abolished pacemaking. However, only blocking NALCN, not TRPC3, degraded burst discharges. Furthermore, local glutamate uncaging readily induced burst discharges within the PDCs, compared with other parts of the neuron, and NALCN channel inhibition dissipated burst generation, indicating the importance of NALCN to the high excitability of PDCs. Therefore, we conclude that PDCs serve as a common base for tonic and burst firing in nigral DA neurons. KEY POINTS: Midbrain dopamine (DA) neurons are slow pacemakers that can generate tonic and burst firings, and the highly excitable proximal dendritic compartments (PDCs) and two Na+ -permeable leak channels, TRPC3 and NALCN, play a key role in pacemaking. We find that slow tonic firing depends on the basal activity of both the NALCN and TRPC3 channels, but that burst firing does not require TRPC3 channels but relies only on NALCN channels. We find that TRPC3 is ubiquitously expressed in the entire somatodendritic compartment, but that NALCN exists only within the PDCs in nigral DA neurons. We show that NALCN channel localization confers high excitability on PDCs and is essential for burst generation in nigral DA neurons. These results suggest that PDCs serve as a common base for tonic and burst firing in nigral DA neurons.

KATP channels as ROS-dependent modulator of neurotransmitter release at the neuromuscular junctions

AimsNeurotransmitter release requires high energy demands, making the nerve terminals metabolically fragile and susceptible to oxidative stress. ATP-sensitive potassium (KATP) channels can be an important regulator orchestrating the influence of metabolic-related signals on exocytosis. Here, the relevance of ROS in KATP channel-dependent control of neurotransmitter release at the frog neuromuscular junction was studied. MethodsMicroelectrode recordings of end plate potentials at the distal and proximal compartments of nerve terminals as well as fluorescent techniques were used. Key findingsActivation of KATP channels in the proximal region suppressed evoked and spontaneous release in a lipid raft-dependent manner. Activation of KATP channels in the distal region reduced solely evoked release which was preserved after lipid raft disruption. Chelation of ROS potentiated the effects of KATP channel activation and unmasked the effects of KATP channel blocker on evoked exocytosis. Activation or inhibition of KATP channels suppressed or enhanced the depressant action of extracellular adenosine on evoked exocytosis. This was accompanied with an increase or decrease in adenosine-induced ROS production, respectively. KATP channel-dependent modulation of adenosine action was halted by antioxidant and NADPH-oxidase inhibitor. Also, activation of KATP channels led to an increase in ROS production suppressing the negative effects of extracellular ATP on evoked release in a ROS-dependent manner. SignificanceKATP channel-mediated modulation of release has specific features in distal and proximal compartments and depends on endogenous ROS levels and lipid raft integrity. Activation of KATP channels suppresses the action of extracellular adenosine and ATP on evoked release by increasing ROS production.

Complex developmental and transcriptional dynamics underlie pollinator-driven evolutionary transitions in nectar spur morphology in Aquilegia (columbine).

Determining the developmental programs underlying morphological variation is key to elucidating the evolutionary processes that generated the stunning biodiversity of the angiosperms. Here, we characterized the developmental and transcriptional dynamics of the elaborate petal nectar spur of Aquilegia (columbine) in species with contrasting pollination syndromes and spur morphologies. We collected petal epidermal cell number and length data across four Aquilegia species, two with short, curved nectar spurs of the bee-pollination syndrome and two with long, straight spurs of the hummingbird-pollination syndrome. We also performed RNA-seq on A. brevistyla (bee) and A. canadensis (hummingbird) distal and proximal spur compartments at multiple developmental stages. Finally, we intersected these data sets with a previous QTL mapping study on spur length and shape to identify new candidate loci. The differential growth between the proximal and distal surfaces of curved spurs is primarily driven by differential cell division. However, independent transitions to straight spurs in the hummingbird syndrome have evolved by increasing differential cell elongation between spur surfaces. The RNA-seq data reveal these tissues to be transcriptionally distinct and point to auxin signaling as being involved with the differential cell elongation responsible for the evolution of straight spurs. We identify several promising candidate genes for future study. Our study, taken together with previous work in Aquilegia, reveals the complexity of the developmental mechanisms underlying trait variation in this system. The framework we established here will lead to exciting future work examining candidate genes and processes involved in the rapid radiation of the genus.

BDNF-dependent modulation of axonal transport is selectively impaired in ALS

Axonal transport ensures long-range delivery of essential cargoes between proximal and distal compartments, and is needed for neuronal development, function, and survival. Deficits in axonal transport have been detected at pre-symptomatic stages in the SOD1G93A and TDP-43M337V mouse models of amyotrophic lateral sclerosis (ALS), suggesting that impairments in this critical process are fundamental for disease pathogenesis. Strikingly, in ALS, fast motor neurons (FMNs) degenerate first whereas slow motor neurons (SMNs) are more resistant, and this is a currently unexplained phenomenon. The main aim of this investigation was to determine the effects of brain-derived neurotrophic factor (BDNF) on in vivo axonal transport in different α-motor neuron (MN) subtypes in wild-type (WT) and SOD1G93A mice. We report that despite displaying similar basal transport speeds, stimulation of wild-type MNs with BDNF enhances in vivo trafficking of signalling endosomes specifically in FMNs. This BDNF-mediated enhancement of transport was also observed in primary ventral horn neuronal cultures. However, FMNs display selective impairment of axonal transport in vivo in symptomatic SOD1G93A mice, and are refractory to BDNF stimulation, a phenotype that was also observed in primary embryonic SOD1G93A neurons. Furthermore, symptomatic SOD1G93A mice display upregulation of the classical non-pro-survival truncated TrkB and p75NTR receptors in muscles, sciatic nerves, and Schwann cells. Altogether, these data indicate that cell- and non-cell autonomous BDNF signalling is impaired in SOD1G93A MNs, thus identifying a new key deficit in ALS.

Temporal and spatial changes in bone mineral content and mechanical properties during breast-cancer bone metastases

Cancer cells favour migration and metastasis to bone tissue for 70–80 % of advanced breast cancer patients and it has been proposed that bone tissue provides attractive physical properties that facilitate tumour invasion, resulting in osteolytic and or osteoblastic metastasis. However, it is not yet known how specific bone tissue composition is associated with tumour invasion. In particular, how compositional and nano-mechanical properties of bone tissue evolve during metastasis, and where in the bone they arise, may affect the overall aggressiveness of tumour invasion, but this is not well understood. The objective of this study is to develop an advanced understanding of temporal and spatial changes in nano-mechanical properties and composition of bone tissue during metastasis. Primary mammary tumours were induced by inoculation of immune-competent BALB/c mice with 4T1 breast cancer cells in the mammary fat pad local to the right femur. Microcomputed tomography and nanoindentation were conducted to quantify cortical and trabecular bone matrix mineralisation and nano-mechanical properties. Analysis was performed in proximal and distal femur regions (spatial analysis) of tumour-adjacent (ipsilateral) and contralateral femurs after 3 weeks and 6 weeks of tumour and metastasis development (temporal analysis). By 3 weeks post-inoculation there was no significant difference in bone volume fraction or nano-mechanical properties of bone tissue between the metastatic femora and healthy controls. However, early osteolysis was indicated by trabecular thinning in the distal and proximal trabecular compartment of tumour-bearing femora. Moreover, cortical thickness was significantly increased in the distal region, and the mean mineral density was significantly higher in cortical and trabecular bone tissue in both proximal and distal regions, of ipsilateral (tumour-bearing) femurs compared to healthy controls. By 6 weeks post-inoculation, overt osteolytic lesions were identified in all ipsilateral metastatic femora, but also in two of four contralateral femora of tumour-bearing mice. Bone volume fraction, cortical area, cortical and trabecular thickness were all significantly decreased in metastatic femora (both ipsilateral and contralateral). Trabecular bone tissue stiffness in the proximal femur decreased in the ipsilateral femurs compared to contralateral and control sites. Temporal and spatial analysis of bone nano-mechanical properties and mineralisation during breast cancer invasion reveals changes in bone tissue composition prior to and following overt metastatic osteolysis, local and distant from the primary tumour site. These changes may alter the mechanical environment of both the bone and tumour cells, and thereby play a role in perpetuating the cancer vicious cycle during breast cancer metastasis to bone tissue.

Measurement of the vaginal wall thickness by transabdominal and transvaginal ultrasound of women with vaginal laxity: a cross-sectional study.

An objective diagnostic method to understand vaginal laxity (VL) is still missing. The aim of our study is to determine whether vaginal wall thickness (VWT) measured by ultrasound may differ according to the abdominal or vaginal techniques and to assess whether clinical variables are associated with vaginal measurements of women with VL. A cross-sectional study conducted at a tertiary hospital included 82 women aged ≥ 18 years with VL complaints assessed by the Vaginal Laxity Questionnaire. Women who reported severe comorbidities or vulvovaginal disorders, previous treatment for VL, and use of vaginal estrogen in the last 6 months were excluded. Participants reporting VL underwent transabdominal (TAUS) and transvaginal ultrasound (TVUS) and physical examination and answered validated questionnaires. Descriptive data were given as mean and standard deviation, median (range), and absolute and relative frequency. The significance level adopted for this study was 5%. Sample size calculation was not performed for the present study. Mean age was 41.20 ± 8.64 years, and most participants were multiparous, with previous vaginal delivery and having vaginal intercourse. A statistically significant difference (up to 3 mm) between TAUS and TVUS measurements of the VWT was found in the proximal, middle-third, and distal compartments. A significant correlation was found between VWT and TAUS or TVUS in the mid-third and distal compartments. A significant correlation was found between the VWT measurements in TVUS and TAUS. Our findings might give the health professional more possibilities for investigating VWT according to patient characteristics.