The effect of metformin combined with liraglutide on gut microbiota of Chinese patients with type 2 diabetes.

BACKGROUND: Metformin (MET) is a first-line therapy for type-2 diabetes mellitus (T2DM). Liraglutide (LRG) is a glucagon-like peptide-1 receptor agonist used as a second-line therapy in combination with MET. METHODS: We performed a longitudinal analysis comparing the gut microbiota of overweight and/or pre-diabetic participants (NCP group) with that of each following their progression to T2DM diagnosis (UNT group) using 16S ribosomal RNA gene sequencing of fecal bacteria samples. We also examined the effects of MET (MET group) and MET plus LRG (MET+LRG group) on the gut microbiota of these participants following 60 days of anti-diabetic drug therapy in two parallel treatment arms. RESULTS: In the UNT group, the relative abundances of Paraprevotella (P = 0.002) and Megamonas (P = 0.029) were greater, and that of Lachnospira (P = 0.003) was lower, compared with the NCP group. In the MET group, the relative abundance of Bacteroides (P = 0.039) was greater, and those of Paraprevotella (P = 0.018), Blautia (P = 0.001), and Faecalibacterium (P = 0.005) were lower, compared with the UNT group. In the MET+LRG group, the relative abundances of Blautia (P = 0.005) and Dialister (P = 0.045) were significantly lower than in the UNT group. The relative abundance of Megasphaera in the MET group was significantly greater than in the MET+LRG group (P = 0.041). CONCLUSIONS: Treatment with MET and MET+LRG results in significant alterations in gut microbiota, compared with the profiles of patients at the time of T2DM diagnosis. These alterations differed significantly between the MET and MET+LRG groups, which suggests that LRG exerted an additive effect on the composition of gut microbiota.

A long non-coding RNA essential for early embryonic development improves somatic cell nuclear transfer somatic cell nuclear transfer efficiency in goats.

In brief: Early embryonic development in goats is a complex and an important process. This study identified a novel long non-coding RNA (lncRNA), lncRNA3720, that appears to affect early embryonic development in goats through histone variants. Abstract: Although abundant lncRNAs have been found to be highly expressed in early embryos, the functions and mechanisms of most lncRNAs in regulating embryonic development remain unclear. This study was conducted to identify the key lncRNAs during embryonic genome activation (EGA) for promoting embryonic development after somatic cell nuclear transfer (SCNT) in goats. We screened and characterized lncRNAs from transcriptome data of in vitro-fertilized, two-cell (IVF-2c) and eight-cell embryos (IVF-8c) and eight-cell SCNT embryos (SCNT-8c). We obtained 12 differentially expressed lncRNAs that were highly expressed in IVF-8c embryos compared to IVF-2c and less expressed in SCNT-8c embryos. After target gene prediction, expression verification, and functional deletion experiments, we found that the expression level of lncRNA3720 affected the early embryonic development in goats. We cloned full-length lncRNA3720 and over-expressed it in goat fetal fibroblasts (GFFs). We identified histone variants by analyzing the transcriptome data from both GFFs and embryos. Gene annotation of the gene library and the literature search revealed that histone variants may have important roles in early embryo development, so we selected them as the potential target genes for lncRNA3720. Lastly, we compensated for the low expression of lncRNA3720 in SCNT embryos by microinjection and showed that the development rate and quality of SCNT embryos were significantly improved. We speculate that lncRNA3720 is a key promoter of embryonic development in goats by interacting with histone variants.

Facile Fabrication of Energetic Nanocomposite Materials by Polydopamine.

Polydopamine-based materials have been widely investigated for incorporation in energetic nanocomposites due to their outstanding adherence. However, these materials are often prepared in alkaline environments, which negatively affects Al nanoparticles. In this study, a one-pot assembly was devised for the preparation of a polydopamine-based Al/CuO energetic nanocomposite material (Al/PDA/CuO) in a neutral environment. The CuO and Al nanoparticles of the Al/PDA/CuO nanothermite were uniformly dispersed and closely combined. Consequently, the Al/PDA/CuO nanothermite was able to release more heat (2069.7 J/g) than physically mixed Al/CuO (1438.9 J/g). Furthermore, the universality of using polydopamine in the assembly of different types of energetic nanocomposite materials was verified, including an organic energetic material-nanothermit (HMX/PDA/Al/CuO nanothermite) and an inorganic oxidant-metal nanocatalyst (AP/PDA/Fe2O3). This study provides a promising route for the preparation of polydopamine-based energetic nanocomposites in neutral aqueous solutions.

DNMT1 facilitates growth of breast cancer by inducing MEG3 hyper-methylation.

BACKGROUND: To understand the effect of DNMT1-mediated MEG3 promoter methylation on breast cancer progression. METHODS: Expression of DNMT1, MEG3 and miR-494-3p was assayed by qRT-PCR and western blot. Methylation-specific PCR was used to examine MEG3 promoter methylation level. ChIP, RNA binding protein immunoprecipitation assay and dual-luciferase reporter gene assay were applied to verify interaction between DNMT1 and MEG3, miR-494-3p and MEG3 and OTUD4. CCK-8, wound healing and Transwell assays were used to detect biological functions of breast cancer cells. Tumor growth was observed by tumor xenograft model. RESULTS: DNMT1 and miR-494-3p were highly expressed while MEG3 and OTUD4 were lowly expressed in breast cancer cells. Knockdown of DNMT1 inhibited progression of breast cancer cells by enhance MEG3 expression through demethylation. MEG3 could downregulate miR-494-3p expression, and OTUD4 was a target of miR-494-3p. Upregulation of MEG3 and downregulation of miR-494-3p both inhibited malignant behavior of cells in vitro. In addition, high MEG3 expression restrained growth of breast cancer in vivo. CONCLUSION: Briefly, our results demonstrated that, DNMT1 induced methylation of MEG3 promoter, and played a key role in breast cancer growth throughmiR-494-3p/OTUD4 axis. These findings provide new insights into molecular therapeutic targets for breast cancer.

Peptide Assembly of Al/CuO Nanothermite for Enhanced Reactivity of Nanoaluminum Particles.

Biological self-assembly procedures, which are generally carried out in an aqueous solution, have been found to be the most promising method for directing the fabrication of diverse nanothermites, including Al/CuO nanothermite. However, the aqueous environment in which Al nanoparticles self-assemble has an impact on their stability. We show that using a peptide to self-assemble Al or CuO nanoparticles considerably improves their durability in phosphate buffer aqueous solution, with Al and CuO nanoparticles remaining intact in aqueous solution for over 2 weeks with minimal changes in the structure. When peptide-assembled Al/CuO nanothermite was compared with a physically mixed sample in phosphate buffer for 30 min, the energy release of the former was higher by 26%. Furthermore, the energy release of peptide-assembled Al/CuO nanocomposite in phosphate buffer showed a 6% reduction by Day 7, while that of the peptide-assembled Al/CuO nanocomposite in ultrapure water was reduced by 75%. Taken together, our study provides an easy method for keeping the thermal activity of Al/CuO nanothermite assembled in aqueous solution.

Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein.

BACKGROUND: Fluorinases play a unique role in the production of fluorine-containing organic molecules by biological methods. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates. RESULTS: A gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused fluorinase was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase displayed on the surface showed good stability while retaining the catalytic activity. The engineered E.coli with surface-displayed fluorinase could be cultured to obtain a larger cell density, which was beneficial for industrial application. And 55% yield of 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-L-methionine (SAM) was achieved by using the whole-cell catalyst. CONCLUSIONS: Here, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of E.coli and maintained its catalytic activity. The surface display provides a new solution for the industrial application of biological fluorination.

Plasma periostin as a biomarker of osteoporosis in postmenopausal women with type 2 diabetes.

INTRODUCTION: Periostin, as an emerging biomarker, is involved in multiple steps in bone metabolism. This study aimed to investigate the correlation between periostin levels and bone mineral density as well as bone turnover markers in postmenopausal women with type 2 diabetes (T2DM). MATERIALS AND METHODS: This study was a cross-sectional study that included 164 postmenopausal women with T2DM as study subjects and 32 age-matched nondiabetic postmenopausal women with normal bone mineral density (BMD) as healthy control subjects. A total of 164 subjects with T2DM were then divided into three groups according to BMD: the normal BMD group (n = 29), the osteopenia group (n = 70), and the osteoporosis group (n = 65). The clinical data of all subjects along with the relevant biochemical parameter data were collected. Plasma periostin was detected using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Plasma periostin levels were significantly increased in T2DM patients with normal BMD compared with healthy controls (p < 0.05). In the diabetic group, plasma periostin levels were significantly elevated with decreased BMD, were positively correlated with osteocalcin levels (r = 0.162, p = 0.039) and were inversely associated with femoral neck BMD (r = - 0.308, p < 0.001) and total femur BMD (r = - 0.295, p < 0.001). In the case of chronic complications, periostin levels were slightly increased in individuals with complications of diabetic retinopathy, diabetic nephropathy and fracture (p > 0.05). CONCLUSIONS: The current study demonstrated that plasma periostin levels were significantly associated with BMD in patients with T2DM, and periostin might act as a novel biochemical marker of osteoporosis in postmenopausal women with type 2 diabetes.

Contribution of Sensory Encoding to Measured Bias.

Signal detection theory (SDT) is a widely used theoretical framework that describes how variable sensory signals are integrated with a decision criterion to support perceptual decision-making. SDT provides two key measurements: sensitivity (d') and bias (c), which reflect the separability of decision variable distributions (signal and noise) and the position of the decision criterion relative to optimal, respectively. Although changes in the subject's decision criterion can be reflected in changes in bias, decision criterion placement is not the sole contributor to measured bias. Indeed, neuronal representations of bias have been observed in sensory areas, suggesting that some changes in bias are because of effects on sensory encoding. To directly test whether the sensory encoding process can influence bias, we optogenetically manipulated neuronal excitability in primary visual cortex (V1) in mice of both sexes during either an orientation discrimination or a contrast detection task. Increasing excitability in V1 significantly decreased behavioral bias, whereas decreasing excitability had the opposite effect. To determine whether this change in bias is consistent with effects on sensory encoding, we made extracellular recordings from V1 neurons in passively viewing mice. Indeed, we found that optogenetic manipulation of excitability shifted the neuronal bias in the same direction as the behavioral bias. Moreover, manipulating the quality of V1 encoding by changing stimulus contrast or interstimulus interval also resulted in consistent changes in both behavioral and neuronal bias. Thus, changes in sensory encoding are sufficient to drive changes in bias measured using SDT.SIGNIFICANCE STATEMENT Perceptual decision-making involves sensory integration followed by application of a cognitive criterion. Using signal detection theory, one can extract features of the underlying decision variables and rule: sensitivity (d') and bias (c). Because bias is measured as the difference between the optimal and actual criterion, it is sensitive to both the sensory encoding processes and the placement of the decision criterion. Here, we use behavioral and electrophysiological approaches to demonstrate that measures of bias depend on sensory processes. Optogenetic manipulations of V1 in mice bidirectionally affect both behavioral and neuronal measures of bias with little effect on sensitivity. Thus, changes in sensory encoding influence bias, and the absence of changes in sensitivity do not preclude changes in sensory encoding.

Neuronal Adaptation Reveals a Suboptimal Decoding of Orientation Tuned Populations in the Mouse Visual Cortex.

Sensory information is encoded by populations of cortical neurons. Yet, it is unknown how this information is used for even simple perceptual choices such as discriminating orientation. To determine the computation underlying this perceptual choice, we took advantage of the robust visual adaptation in mouse primary visual cortex (V1). We first designed a stimulus paradigm in which we could vary the degree of neuronal adaptation measured in V1 during an orientation discrimination task. We then determined how adaptation affects task performance for mice of both sexes and tested which neuronal computations are most consistent with the behavioral results given the adapted population responses in V1. Despite increasing the reliability of the population representation of orientation among neurons, and improving the ability of a variety of optimal decoders to discriminate target from distractor orientations, adaptation increases animals' behavioral thresholds. Decoding the animals' choice from neuronal activity revealed that this unexpected effect on behavior could be explained by an overreliance of the perceptual choice circuit on target preferring neurons and a failure to appropriately discount the activity of neurons that prefer the distractor. Consistent with this all-positive computation, we find that animals' task performance is susceptible to subtle perturbations of distractor orientation and optogenetic suppression of neuronal activity in V1. This suggests that to solve this task the circuit has adopted a suboptimal and task-specific computation that discards important task-related information.SIGNIFICANCE STATEMENT A major goal in systems neuroscience is to understand how sensory signals are used to guide behavior. This requires determining what information in sensory cortical areas is used, and how it is combined, by downstream perceptual choice circuits. Here we demonstrate that when performing a go/no-go orientation discrimination task, mice suboptimally integrate signals from orientation tuned visual cortical neurons. While they appropriately positively weight target-preferring neurons, they fail to negatively weight distractor-preferring neurons. We propose that this all-positive computation may be adopted because of its simple learning rules and faster processing, and may be a common approach to perceptual decision-making when task conditions allow.

Magnitude, time course, and specificity of rapid adaptation across mouse visual areas.

Adaptation is a ubiquitous feature of sensory processing whereby recent experience shapes future responses. The mouse primary visual cortex (V1) is particularly sensitive to recent experience, where a brief stimulus can suppress subsequent responses for seconds. This rapid adaptation profoundly impacts perception, suggesting that its effects are propagated along the visual hierarchy. To understand how rapid adaptation influences sensory processing, we measured its effects at key nodes in the visual system: in V1, three higher visual areas (HVAs: lateromedial, anterolateral, and posteromedial), and the superior colliculus (SC) in awake mice of both sexes using single-unit recordings. Consistent with the feed-forward propagation of adaptation along the visual hierarchy, we find that neurons in layer 4 adapt less strongly than those in other layers of V1. Furthermore, neurons in the HVAs adapt more strongly, and recover more slowly, than those in V1. The magnitude and time course of adaptation was comparable in each of the HVAs and in the SC, suggesting that adaptation may not linearly accumulate along the feed-forward visual processing hierarchy. Despite the increase in adaptation in the HVAs compared with V1, the effects were similarly orientation specific across all areas. These data reveal that adaptation profoundly shapes cortical processing, with increasing impact at higher levels in the cortical hierarchy, and also strongly influencing computations in the SC. Thus, we find robust, brain-wide effects of rapid adaptation on sensory processing.NEW & NOTEWORTHY Rapid adaptation dynamically alters sensory signals to account for recent experience. To understand how adaptation affects sensory processing and perception, we must determine how it impacts the diverse set of cortical and subcortical areas along the hierarchy of the mouse visual system. We find that rapid adaptation strongly impacts neurons in primary visual cortex, the higher visual areas, and the colliculus, consistent with its profound effects on behavior.

Apical PtdIns(4,5)P2 is required for ciliogenesis and suppression of polycystic kidney disease.

Cilia are hair-like structures that function like antennae to detect chemical and mechanical signals in the environment. Recently, phosphoinositides were shown to play an important role in cilia assembly and disassembly. However, the precise molecular and cellular mechanisms underlying this process remain unknown. Here, we report that suppression of apical phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P2], by overexpressing apically targeted PtdIns(4,5)P2 phosphatase or by knocking down type I phosphatidylinositol 4-phosphate 5-kinase (Pip5k1), leads to ciliogenesis defects and polycystic kidney disease (PKD) in zebrafish embryos that phenocopied inpp5e mutant, a Joubert syndrome model. We further demonstrate that decreased expression of apical PtdIns(4,5)P2 disrupted apical ezrin recruitment, F-actin organization, and basal body docking. Moreover, the ciliogenesis and polycystic kidney defects in PtdIns(4,5)P2-depleted embryos can be rescued by overexpression of ezrin. Finally, Pip5k1a overexpression rescued the ciliogenesis defects and PKD phenotypes in Inpp5e-depleted embryos. Taken together, our results reveal that apical PtdIns(4,5)P2 is essential for ciliogenesis and the prevention of PKD and suggest a novel possibility for treating PKD and other human ciliopathies.-Xu, W., Jin, M., Huang, W., Wang, H., Hu, R., Li, J., Cao, Y. Apical PtdIns(4,5)P2 is required for ciliogenesis and suppression of polycystic kidney disease.

Mutagenesis of putative ciliary genes with the CRISPR/Cas9 system in zebrafish identifies genes required for retinal development.

Cilia are conserved microtubule-based organelles that function as mechanical and chemical sensors in various cell types. By bioinformatic, genomic, and proteomic studies, more than 2000 proteins have been identified as cilium-associated proteins or putative ciliary proteins; these proteins are referred to as the ciliary proteome or the ciliome. However, little is known about the function of these numerous putative ciliary proteins in cilia. To identify the possible new functional proteins or pathways in cilia, we carried out a small-scale genetic screen targeting 54 putative ciliary genes by using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. We successfully constructed 54 zebrafish mutants, and 8 of them displayed microphthalmias. Three of these 8 genes encode proteins for protein transport, suggesting the important roles of protein transport in retinal development. In situ hybridization revealed that all these genes are expressed in zebrafish eyes. Furthermore, polo-like kinase 1 was required for ciliogenesis in neural tube. We uncovered the potential function of the ciliary genes for the retinal development of zebrafish.-Hu, R., Huang, W., Liu, J., Jin, M., Wu, Y., Li, J., Wang, J., Yu, Z., Wang, H., Cao, Y. Mutagenesis of putative ciliary genes with the CRISPR/Cas9 system in zebrafish identifies genes required for retinal development.

Safety assessment and antioxidant evaluation of betulin by LC-MS combined with free radical assays.

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

A high-throughput metabolomics approach for the comprehensive differentiation of four Pulsatilla Adans herbs combined with a nontargeted bidirectional screen for rapid identification of triterpenoid saponins.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

Study on the metabolites of betulinic acid in vivo and in vitro by ultra high performance liquid chromatography with time-of-flight mass spectrometry.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

Triphenyltin exposure affects mating behaviors and attractiveness to females during mating in male guppies (Poecilia reticulata).

The impacts of triphenyltin (TPT) on ecological health have been of great concern due to their widespread use and ubiquity in aquatic ecosystems. However, little is known about the effects of TPT on the reproductive behaviors of fishes. Therefore, the present study was conducted to investigate the effects of TPT at environmentally relevant concentrations (0, 1 and 10 ng Sn/L) on the mating behaviors and the attractiveness to females during mating in male guppies (Poecilia reticulata). The results showed that TPT exposure disturbed the mating behaviors; the TPT-exposed male fish performed more sneaking attempts, but no changes in sigmoid courtship were displayed. The increases in sneaking attempts might be related to increases in testosterone levels induced by TPT exposure. In the context of a competing male, the TPT-exposed males were less attractive to females during mating. The decreases in attractiveness might be related to decreases in carotenoid-based coloration, shown as decreases in caudal fin redness values and skin carotenoid contents. In addition, TPT-induced total antioxidant capacities, the activities of superoxide dismutase and catalase, and the contents of malondialdehyde in liver and intestinal tissues indicated increases in oxidative stress. Both oxidative stress and coloration are linked to carotenoids. Thus, we speculated that the TPT-exposed males might use carotenoids to cope with increases in oxidative stress at the expense of carotenoid-based coloration. The disruption of mating behaviors and the decrease in attractiveness to females in male fish could result in reproductive failure. The present study underscores the importance of using behavioral tests as a sensitive tool in assessing the impact of pollutants present in aquatic environments.

Reconfigurable logic in nanosecond Cu/GeTe/TiN filamentary memristors for energy-efficient in-memory computing.

Owing to the capability of integrating the information storage and computing in the same physical location, in-memory computing with memristors has become a research hotspot as a promising route for non von Neumann architecture. However, it is still a challenge to develop high performance devices as well as optimized logic methodologies to realize energy-efficient computing. Herein, filamentary Cu/GeTe/TiN memristor is reported to show satisfactory properties with nanosecond switching speed (<60 ns), low voltage operation (<2 V), high endurance (>104 cycles) and good retention (>104 s @85 °C). It is revealed that the charge carrier conduction mechanisms in high resistance and low resistance states are Schottky emission and hopping transport between the adjacent Cu clusters, respectively, based on the analysis of current-voltage behaviors and resistance-temperature characteristics. An intuitive picture is given to describe the dynamic processes of resistive switching. Moreover, based on the basic material implication (IMP) logic circuit, we proposed a reconfigurable logic method and experimentally implemented IMP, NOT, OR, and COPY logic functions. Design of a one-bit full adder with reduction in computational sequences and its validation in simulation further demonstrate the potential practical application. The results provide important progress towards understanding of resistive switching mechanism and realization of energy-efficient in-memory computing architecture.

The Joubert Syndrome Protein Inpp5e Controls Ciliogenesis by Regulating Phosphoinositides at the Apical Membrane.

Phosphoinositides, a family of phosphorylated derivatives of phosphatidylinositol (PtdIns), are tightly regulated both temporally and spatially by PtdIns phosphatases and kinases. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause Joubert syndrome, a human disorder associated with numerous ciliopathic defects, including renal cyst formation, linking phosphoinositides to ciliopathies. However, the molecular mechanism by which INPP5E-mediated PtdIns signaling regulates ciliogenesis and cystogenesis is unclear. Here, we utilized an in vivo vertebrate model of renal cystogenesis to show that Inpp5e enzymatic activity at the apical membrane directs apical docking of basal bodies in renal epithelia. Knockdown or knockout of inpp5e led to ciliogenesis defects and cystic kidneys in zebrafish. Furthermore, knockdown of inpp5e in embryos led to defects in cell polarity, cortical organization of F-actin, and apical segregation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 Knockdown of the ezrin gene, which encodes an ezrin/radixin/moesin (ERM) protein that crosslinks PtdIns(4,5)P2 and F-actin, phenocopied inpp5e knockdowns. Notably, overexpression of the ezrin gene rescued inpp5e morphants. Finally, treatment with the PI 3-kinase inhibitor LY294002, which decreases PtdIns(3,4,5)P3 levels, rescued the cellular, phenotypic, and renal functional defects in inpp5e-knockdown embryos. Together, our data indicate that Inpp5e functions as a key regulator of cell polarity in the renal epithelia, by inhibiting PtdIns(3,4,5)P3 and subsequently stabilizing PtdIns(4,5)P2 and recruiting Ezrin, F-actin, and basal bodies to the apical membrane, and suggest a possible novel approach for treating human ciliopathies.

UPLC-Q-TOF-MS/MS-guided dereplication of Pulsatilla chinensis to identify triterpenoid saponins.

INTRODUCTION: Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. OBJECTIVE: To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. METHODOLOGY: In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. RESULTS: As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. CONCLUSION: This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

Simultaneous determination of 12 active components in the roots of Pulsatilla chinensis using tissue-smashing extraction with liquid chromatography and mass spectrometry.

Ultra high performance liquid chromatography coupled with mass spectrometry and combining a tissue-smashing extraction technique was developed for the simultaneous quantitative analysis of 12 compounds in the roots of Pulsatilla chinensis. Among them, compound 6 was characterized and accurately quantified in this herb for the first time. The parameters of extraction condition were simultaneously optimized with a Box-Behnken design and Derringer's function. The optimized conditions were as follows: sample quantity of 0.5 g, ethanol concentration of 70%, and extraction time of 200 s. Multiple-reaction monitoring scanning was employed for the quantification between positive and negative mode in a single run of 6 min. Full validation of the method was carried out, and the results indicated that the method was rapid, specific, and reliable. The developed method was successfully applied to quantify the 12 compounds in 33 batches of P. chinensis from different provinces. Moreover, the principal component analysis was performed to compare the P. chinensis collected from different provinces of China based on quantitative data and the results indicated that the content of compounds could be used to differentiate the origins of P. chinensis. These results demonstrated that this method is feasible and reliable for the quality control of P. chinensis.