Physical Techniques to Remove Residual Stone Fragments in the Urinary System.

BACKGROUND: Although significant progress has been made in treatment techniques for renal and ureteral calculi, residual fragments (RF) persisting long after treatment pose a serious threat to patients' health. A variety of novel physical techniques and extraction devices are currently being developed to promote the removal of RF from the urinary system, and a series of in vivo experiments have demonstrated their safety and efficacy. SUMMARY: External physical vibration lithecbole, magnetic extraction, biocompatible stone adhesive-based methods, and ultrasonic propulsion technologies are examples of innovative therapies that can promote the clearance of RF and improve the stone-free rate. In conclusion, the physical treatment of these RF needs to be optimized and improved. They are a promising technique for improving the efficiency of endovascular urology, and further in vivo studies are needed to confirm their safety and efficacy. KEY MESSAGES: We have summarized the literature on removal of RF by physical methods in recent years, especially the new progress.

A high-throughput 3D cantilever array to model airway smooth muscle hypercontractility in asthma.

Asthma is often characterized by tissue-level mechanical phenotypes that include remodeling of the airway and an increase in airway tightening, driven by the underlying smooth muscle. Existing therapies only provide symptom relief and do not improve the baseline narrowing of the airway or halt progression of the disease. To investigate such targeted therapeutics, there is a need for models that can recapitulate the 3D environment present in this tissue, provide phenotypic readouts of contractility, and be easily integrated into existing assay plate designs and laboratory automation used in drug discovery campaigns. To address this, we have developed DEFLCT, a high-throughput plate insert that can be paired with standard labware to easily generate high quantities of microscale tissues in vitro for screening applications. Using this platform, we exposed primary human airway smooth muscle cell-derived microtissues to a panel of six inflammatory cytokines present in the asthmatic niche, identifying TGF-ß1 and IL-13 as inducers of a hypercontractile phenotype. RNAseq analysis further demonstrated enrichment of contractile and remodeling-relevant pathways in TGF-ß1 and IL-13 treated tissues as well as pathways generally associated with asthma. Screening of 78 kinase inhibitors on TGF-ß1 treated tissues suggests that inhibition of protein kinase C and mTOR/Akt signaling can prevent this hypercontractile phenotype from emerging, while direct inhibition of myosin light chain kinase does not. Taken together, these data establish a disease-relevant 3D tissue model for the asthmatic airway, which combines niche specific inflammatory cues and complex mechanical readouts that can be utilized in drug discovery efforts.

A cell-level quality control workflow for high-throughput image analysis.

BACKGROUND: Image-based high throughput (HT) screening provides a rich source of information on dynamic cellular response to external perturbations. The large quantity of data generated necessitates computer-aided quality control (QC) methodologies to flag imaging and staining artifacts. Existing image- or patch-level QC methods require separate thresholds to be simultaneously tuned for each image quality metric used, and also struggle to distinguish between artifacts and valid cellular phenotypes. As a result, extensive time and effort must be spent on per-assay QC feature thresholding, and valid images and phenotypes may be discarded while image- and cell-level artifacts go undetected. RESULTS: We present a novel cell-level QC workflow built on machine learning approaches for classifying artifacts from HT image data. First, a phenotype sampler based on unlabeled clustering collects a comprehensive subset of cellular phenotypes, requiring only the inspection of a handful of images per phenotype for validity. A set of one-class support vector machines are then trained on each biologically valid image phenotype, and used to classify individual objects in each image as valid cells or artifacts. We apply this workflow to two real-world large-scale HT image datasets and observe that the ratio of artifact to total object area (ARcell) provides a single robust assessment of image quality regardless of the underlying causes of quality issues. Gating on this single intuitive metric, partially contaminated images can be salvaged and highly contaminated images can be excluded before image-level phenotype summary, enabling a more reliable characterization of cellular response dynamics. CONCLUSIONS: Our cell-level QC workflow enables identification of artificial cells created not only by staining or imaging artifacts but also by the limitations of image segmentation algorithms. The single readout ARcell that summaries the ratio of artifacts contained in each image can be used to reliably rank images by quality and more accurately determine QC cutoff thresholds. Machine learning-based cellular phenotype clustering and sampling reduces the amount of manual work required for training example collection. Our QC workflow automatically handles assay-specific phenotypic variations and generalizes to different HT image assays.

Selective DYRK1A Inhibitor for the Treatment of Type 1 Diabetes: Discovery of 6-Azaindole Derivative GNF2133.

Autoimmune deficiency and destruction in either ß-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting ß-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human ß-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).

Tau overexpression exacerbates neuropathology after repeated mild head impacts in male mice.

Repeated mild traumatic brain injury (rmTBI) can lead to development of chronic traumatic encephalopathy (CTE), which is characterized by progressive neurodegeneration with presence of white matter damage, gliosis and hyper-phosphorylated tau. While animal models of rmTBI have been documented, few characterize the molecular pathogenesis and expression profiles of relevant injured brain regions. Additionally, while the usage of transgenic tau mice in rmTBI is prevalent, the effects of tau on pathological outcomes has not been well studied. Here we characterized a 42-impact closed-head rmTBI paradigm on 3-4 month old male C57BL/6 (WT) and Tau-overexpressing mice (Tau58.4). This injury paradigm resulted in chronic gliosis, T-cell infiltration, and demyelination of the optic nerve and associated white matter tracts at 1-month post-injury. At 3-months post-injury, Tau58.4 mice showed progressive neuroinflammation and neurodegeneration in multiple brain regions compared to WT mice. Corresponding to histopathology, RNAseq of the optic nerve tract at 1-month post-injury showed significant upregulation of inflammatory pathways and downregulation of myelin synthetic pathways in both genotypes. However, Tau58.4 mice showed additional changes in neurite development, protein processing, and cell stress. Comparisons with published transcriptomes of human Alzheimer's Disease and CTE revealed common signatures including neuroinflammation and downregulation of protein phosphatases. We next investigated the demyelination and T-cell infiltration phenotypes to determine whether these offer potential avenues for therapeutic intervention. Tau58.4 mice were treated with the histamine H3 receptor antagonist GSK239512 for 1-month post-injury to promote remyelination of white matter lesions. This restored myelin gene expression to sham levels but failed to repair the histopathologic lesions. Likewise, injured T-cell-deficient Rag2/Il2rg (R2G2) mice also showed evidence for inflammation and loss of myelin. However, unlike immune-competent mice, R2G2 mice had altered myeloid cell gene expression and fewer demyelinated lesions. Together this data shows that rmTBI leads to chronic white matter inflammatory demyelination and axonal loss exacerbated by human tau overexpression but suggests that immune-suppression and remyelination alone are insufficient to reverse damage.

Inner membrane fusion mediates spatial distribution of axonal mitochondria.

In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution.

Inhibition of DYRK1A and GSK3B induces human ß-cell proliferation.

Insufficient pancreatic ß-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from ß-cells in diabetic patients, no pharmacological agents have been described that increase ß-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust ß-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces ß-cell proliferation, increases ß-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore ß-cell mass, and highlights a tractable pathway for future drug discovery efforts.

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2.

Kinesin-5 is an essential mitotic motor. However, how its spatial-temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification-tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end-directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end-directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.

A nonmotor microtubule binding site in kinesin-5 is required for filament crosslinking and sliding.

Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.

Lamin B counteracts the kinesin Eg5 to restrain spindle pole separation during spindle assembly.

Lamin B is a component of the membranous spindle matrix isolated from Xenopus egg extracts, and it is required for proper spindle morphogenesis. Besides lamin B, the spindle matrix contains spindle assembly factors (SAFs) such as Eg5 and dynein which are known to regulate microtubule organization and SAFs known to promote microtubule assembly such as Maskin and XMAP215. Because lamin B does not bind directly to microtubules, it must affect spindle morphogenesis indirectly by influencing the function of spindle matrix-associated SAFs. Using different assays in Xenopus egg extracts, we found that depleting lamin B caused formation of elongated and multipolar spindles, which could be reversed by partially inhibiting the kinesin Eg5, revealing an antagonistic relationship between Eg5 and lamin B. However, lamin B only very weakly antagonizes Eg5 in mediating poleward microtubule-flux based on fluorescence speckle microscopy. Depleting lamin B led to a very small but statistically significant increase in flux. Furthermore, flux reduction caused by partial Eg5 inhibition is only slightly reversed by removing lamin B. Because lamin B does not bind to Eg5, our studies suggest two nonexclusive mechanisms by which lamin B can indirectly antagonize Eg5. It could function in a network that restricts Eg5-driven microtubule sliding only when microtubules come into transient contact with the network. Lamin B could also function to sequester microtubule polymerization activities within the spindle. Without lamin B, increased microtubule assembly caused by the released SAFs would lead to excessive microtubule sliding that results in formation of elongated and multipolar spindles.

A new lignan from the seeds of Arctium lappa.

A new compound, neoarctin A (1), together with nine known compounds (2-10), were obtained from the ethanolic extract of the seeds of Arctium lappa. The structure of 1 was elucidated on the basis of spectral and chemical evidence.