Quality assessment of rose tea with different drying methods based on physicochemical properties, HS-SPME-GC-MS, and GC-IMS.

The purpose of this study is to compare the physicochemical properties and volatile flavor compounds of rose tea obtained by the methods of normal temperature drying, hot-air drying (HAD), and vacuum freeze-drying (VFD) and to evaluate the quality of rose tea. The physicochemical results showed that the content of ascorbic acid (VC) and the pH value was the highest in rose tea obtained by HAD. The contents of anthocyanin, proanthocyanidins, and total phenols were highest in rose tea obtained by VFD. However, there was no significant difference in total flavonoids between drying methods. The volatile organic compounds (VOCs) in rose tea with different drying methods were analyzed by headspace solid-phase microextraction-gas chromatography-mass spectroscopy (HS-SPME-GC-MS) and HS GC-ion mobility spectroscopy (HS-GC-IMS), and the flavor fingerprint of rose tea was established by principal component analysis (PCA). The concentration of VOCs in rose tea varied greatly with different drying methods. The main flavor compounds of rose tea were alcohols, esters, aldehydes, and terpenoids. HS-GC-IMS was used for the identification of volatile flavor compounds of rose tea, thereby helping to assess the quality of rose tea. In addition, the rose tea samples with different drying methods were well distinguished by PCA. This study deepens the understanding of the physicochemical properties and volatile flavor compounds of rose tea with different drying methods and provides a reference for the identification of rose tea with different drying methods. PRACTICAL APPLICATION: This study deepens the understanding of the physicochemical properties and volatile flavor compounds of rose tea with different drying methods and provides a reference for the identification of rose tea with different drying methods. It also provides an effective theoretical basis for consumers to buy rose tea.

Bridging therapy and direct mechanical thrombectomy in the treatment of cardiogenic cerebral infarction with anterior circulation macrovascular occlusion.

BACKGROUND: Intravenous thrombolysis is an important treatment for cerebral infarction. However, it is difficult to achieve good results if the patient is complicated with anterior circulation macrovascular occlusion. In addition, the vascular recanalization rate is low, so mechanical thrombectomy, that is, bridging therapy, is needed. AIM: To investigate the efficacy and safety of bridging therapy and direct mechanical thrombectomy in the treatment of cardiogenic cerebral infarction with anterior circulation macrovascular occlusion. METHODS: Ninety-six patients in our hospital with cardiogenic cerebral infarction with anterior circulation macrovascular occlusion from January 2017 to July 2020 were divided into a direct thrombectomy group (n = 48) and a bridging group (n = 48). Direct mechanical thrombectomy was performed in the direct thrombectomy group, and bridging therapy was used in the bridging treatment group. Comparisons were performed for the treatment data of the two groups (from admission to imaging examination, from admission to arterial puncture, from arterial puncture to vascular recanalization, and from admission to vascular recanalization), vascular recanalization rate, National Institutes of Health Stroke Scale (NIHSS) and Glasgow Coma Scale (GCS) scores before and after treatment, prognosis and incidence of adverse events. RESULTS: In the direct thrombectomy group, the time from admission to imaging examination was 24.32 ± 8.61 min, from admission to arterial puncture was 95.56 ± 37.55 min, from arterial puncture to vascular recanalization was 54.29 ± 21.38 min, and from admission to revascularization was 156.88 ± 45.51 min, and the corresponding times in the bridging treatment group were 25.38 ± 9.33 min, 100.45 ± 39.30 min, 58.14 ± 25.56 min, and 161.23 ± 51.15 min; there were no significant differences between groups (P=0.564, 0.535, 0.426, and 0.661, respectively). There was no significant difference in the recanalization rate between the direct thrombectomy group (79.17%) and the bridging group (75.00%) (P = 0.627). There were no significant differences between the direct thrombectomy group (16.69 ± 4.91 and 12.12 ± 2.07) and the bridging group (7.13 ± 1.23 and (14.40 ± 0.59) in preoperative NIHSS score and GCS score (P = 0.200 and 0.203, respectively). After the operation, the NIHSS scores in both groups were lower than those before the operation, and the GCS scores were higher than those before the operation. There was no significant difference in NIHSS and GCS scores between the direct thrombectomy group (6.91 ± 1.10 and 14.19 ± 0.65) and the bridging group (7.13 ± 1.23 and 14.40 ± 0.59) (P = 0.358 and 0.101, respectively). There was no significant difference in the proportion of patients who achieved a good prognosis between the direct thrombectomy group (52.08%) and the bridging group (50.008%) (P = 0.838). There was no significant difference in the incidence of adverse events between the direct thrombectomy group (6.25%) and the bridging group (8.33%) (P = 0.913). CONCLUSION: Bridging therapy and direct mechanical thrombectomy can safely treat cardiogenic cerebral infarction with anterior circulation macrovascular occlusion, achieve good vascular recanalization effects and prognoses, and improve the neurological function of patients.

Antitumor effects and molecular mechanisms of action of natural products in ovarian cancer.

Ovarian cancer is a common malignancy and the second leading cause of mortality among females with genital tract cancer. At present, postoperative platinum drugs and paclitaxel-based chemotherapy is the gold standard treatment for ovarian cancer. However, patients who receive this chemotherapy often develop cumulative toxic effects and are prone to chemotherapy resistance. Therefore, it is necessary to determine more effective treatment options that would be better tolerated by patients. Recent studies have reported the therapeutic effects of numerous natural products in patients with ovarian cancer. Notably, these natural ingredients do not induce adverse effects in healthy cells and tissues, suggesting that natural products may serve as a safe alternative treatment for ovarian cancer. The antitumor effects of natural products are attributed to suppression of cell proliferation and metastasis, stimulation of autophagy, improved chemotherapy sensitivity, and induction of apoptosis. The present review focused on the antitumor effects of several natural products, including curcumin, resveratrol, ginsenosides, (-)-epigallocatechin-3-gallate and quercetin, which are increasingly being investigated as therapeutic options in ovarian cancer, and discussed the molecular mechanisms involved in cell proliferation, apoptosis, autophagy, metastasis and sensitization.

[Genetic diversity of 6 species in Polygonatum by SSR marker].

Polygonatum is a genus of the perennial herb family Liliaceae, with great potential in food, medicine and other field. In this study, genetic diversity and cluster structure analysis of 6 species in Polygonatum were investigated the molecular marker technique of simple sequence repeat (SSR). A total of 49 SSR makers were used to study genetic diversity and population structure within 60 germplasm resources which obtained from 38 counties and cities in 14 provinces of China. A total of 211 alleles were identified and the number of alleles ranged from 2 to 10, with an average of 4.306 1 alleles per SSR. The range of polymorphism information content (PIC) varied from 0.731 8 to 0.031 7, with the average of 0.309 6. The cluster analysis classified 60 germplasm resources into four defined groups at the genetic distance value of 0.26, among which most species with relatives were clustered into the same group. Extraordinarily, there were 6 germplasm resources clustered into other species, indicating that the classification of inter-genus and geographical distribution was crossed in Polygonatum. The genetic diversity index of the 4 geographical populations from high to low was: Western region, Central China, Southern China, Eastern China. The population structure analysis, also indicating divided the entire collection into four groups, which was similar to the assignment pattern of cluster structure analysis. These results suggested that the Polygonatum germplasm resources used in this study is rich in relatively high genetic diversity with large variation range, relatively fuzzy boundaries of species. It appeared the phenomenon that there is a difference decreases between the alternate leaf system and the rotation leaf system. The genetic diversity in the western region was higher than that in other regions, and the western region may be the origin center of the genus Polygonatum.

Molecular overlap in the regulation of SK channels by small molecules and phosphoinositides.

Phosphatidylinositol 4,5-bisphosphate (PIP2) directly interacts with the small-conductance Ca2+-activated K+ 2-a (SK2-a) channel/calmodulin complex, serving as a critical element in the regulation of channel activity. We report that changes of protein conformation in close proximity to the PIP2 binding site induced by a small-molecule SK channel modulator, NS309, can effectively enhance the interaction between the protein and PIP2 to potentiate channel activity. This novel modulation of PIP2 sensitivity by small-molecule drugs is likely not to be limited in its application to SK channels, representing an intriguing strategy to develop drugs controlling the activity of the large number of PIP2-dependent proteins.

Selective phosphorylation modulates the PIP2 sensitivity of the CaM-SK channel complex.

Phosphatidylinositol bisphosphate (PIP2) regulates the activities of many membrane proteins, including ion channels, through direct interactions. However, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we show that PIP2 is a key cofactor for activation of small conductance Ca2+-activated potassium channels (SKs) by Ca(2+)-bound calmodulin (CaM). Removal of the endogenous PIP2 inhibits SKs. The PIP2-binding site resides at the interface of CaM and the SK C terminus. We further demonstrate that the affinity of PIP2 for its target proteins can be regulated by cellular signaling. Phosphorylation of CaM T79, located adjacent to the PIP2-binding site, by casein kinase 2 reduces the affinity of PIP2 for the CaM-SK channel complex by altering the dynamic interactions among amino acid residues surrounding the PIP2-binding site. This effect of CaM phosphorylation promotes greater channel inhibition by G protein-mediated hydrolysis of PIP2.

Unstructured to structured transition of an intrinsically disordered protein peptide in coupling Ca²âº-sensing and SK channel activation.

Most proteins, such as ion channels, form well-organized 3D structures to carry out their specific functions. A typical voltage-gated potassium channel subunit has six transmembrane segments (S1-S6) to form the voltage-sensing domain and the pore domain. Conformational changes of these domains result in opening of the channel pore. Intrinsically disordered (ID) proteins/peptides are considered equally important for the protein functions. However, it is difficult to explore the structural features underlying the functions of ID proteins/peptides by conventional methods, such as X-ray crystallography, because of the flexibility of their secondary structures. Unlike voltage-gated potassium channels, families of small- and intermediate-conductance Ca(2+)-activated potassium (SK/IK) channels with important roles in regulating membrane excitability are activated exclusively by Ca(2+)-bound calmodulin (CaM). Upon binding of Ca(2+) to CaM, a 2 × 2 structure forms between CaM and the CaM-binding domain. A channel fragment that connects S6 and the CaM-binding domain is not visible in the protein crystal structure, suggesting that this fragment is an ID fragment. Here we show that the conformation of the ID fragment in SK channels becomes readily identifiable in the presence of NS309, the most potent compound that potentiates the channel activities. This well-defined conformation of the ID fragment, stabilized by NS309, increases the channel open probability at a given Ca(2+) concentration. Our results demonstrate that the ID fragment, itself a target for drugs modulating SK channel activities, plays a unique role in coupling Ca(2+) sensing by CaM and mechanical opening of SK channels.

Identification of the functional binding pocket for compounds targeting small-conductance Ca²âº-activated potassium channels.

Small- and intermediate-conductance Ca(2+)-activated potassium channels, activated by Ca(2+)-bound calmodulin, have an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potential for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-ethyl-2-benzimidazolinone (1-EBIO) class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class.

Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis.

Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca(2+) channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin-channel complex is Ca(2+) dependent. A glutamate residue, E264, in endophilin is part of the primary Ca(2+) sensor for Ca(2+)-dependent formation of the channel-endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca(2+), and a closed mode in the presence of Ca(2+). Binding of Ca(2+) switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or ß1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of ß1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca(2+) channels in the presence of Ca(2+). Our results show that modal switching by Ca(2+) allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal.

Structural basis for calmodulin as a dynamic calcium sensor.

Calmodulin is a prototypical and versatile Ca(2+) sensor with EF hands as its high-affinity Ca(2+) binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca(2+)-dependent signaling. Upon binding Ca(2+), calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin's ability to bind Ca(2+). Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca(2+)-bound calmodulin but show different sensitivity to Ca(2+) for their activation. Protein crystal structures and other experiments show that, depending on which SK2 splice variant it binds to, calmodulin adopts drastically different conformations with different affinities for Ca(2+) at its C-lobe. Such target protein-induced conformational changes make calmodulin a dynamic Ca(2+) sensor capable of responding to different Ca(2+) concentrations in cellular Ca(2+) signaling.

Structure of the SH3 domain of rat endophilin A2.

The crystal structure of the SH3 domain of rat endophilin A2 has been determined by the multiwavelength anomalous dispersion method and refined at a resolution of 1.70 A to R and R(free) values of 0.196 and 0.217, respectively. The structure adheres to the canonical SH3-domain fold and is highly similar to those of the corresponding domains of endophilins A1 and A3. An intermolecular packing interaction between two molecules in the lattice exploits features that are commonly observed in SH3-domain ligand recognition, including the insertion of a proline side chain into the ligand-binding groove of the protein and the recognition of a basic residue by a cluster of acidic side chains on the RT loop.

Essential role of the LIM domain in the formation of the PKCepsilon-ENH-N-type Ca2+ channel complex.

A LIM domain is a specialized double-zinc finger motif found in a variety of proteins. LIM domains are thought to function as molecular modules, mediating specific protein-protein interactions in cellular signaling. In a recent study, we have demonstrated that ENH, which has three consecutive LIM domains, acts as an adaptor protein for the formation of a functional PKCepsilon-ENH-N-type Ca2+ channel complex in neurons. Formation of this complex selectively recruits PKCepsilon to its specific substrate, N-type Ca2+ channels, and is critical for rapid and efficient potentiation of the Ca2+ channel activity by PKC in neurons. However, it is not clear whether changes in the local Ca2+ concentrations near the channel mouth may affect the formation of the triprotein complex. Furthermore, the molecular determinants for the interactions among these three proteins remain unknown. Biochemical studies were performed to address these questions. Within the physiological Ca2+ concentration range (0-300 microM), binding of ENH to the channel C-terminus was significantly increased by Ca2+, whereas increased Ca2+ levels led to dissociation of PKCepsilon from ENH. Mutagenesis studies revealed that the second LIM domain in ENH was primarily responsible for Ca2+-dependent binding of ENH to both the Ca2+ channel C-terminus and PKCepsilon. ENH existed as a dimer in vivo. PKCepsilon translocation inhibition peptide, which blocks the translocation of PKCepsilon from the cytosol to the membrane, inhibited the interaction between PKCepsilon and ENH. These results provide a molecular mechanism for how the PKCepsilon-ENH-N-type Ca2+ channel complex is formed and regulated, as well as potential drug targets to selectively disrupt the PKC signaling complex.

Modulation of the cardiac sodium channel NaV1.5 by Fyn, a Src family tyrosine kinase.

Dynamic modulation of ion channels can produce dramatic alterations of electrical excitability in cardiac myocytes. This study addresses the effects of the Src family tyrosine kinase Fyn on Na(V)1.5 cardiac sodium channels. Sodium currents were acquired by whole cell recording on HEK-293 cells transiently expressing Na(V)1.5. Acute treatment of cells with insulin caused a depolarizing shift in steady-state inactivation, an effect eliminated by the Src-specific tyrosine kinase inhibitor PP2. Sodium channels were coexpressed with either constitutively active (Fyn(CA)) or catalytically inactive (Fyn(KD)) variants of Fyn. Fyn(CA) caused a 10-mV depolarizing shift of steady-state inactivation compared with Fyn(KD) without altering the activation conductance-voltage relationship. Comparable effects of these Fyn variants were obtained with whole-cell and perforated-patch recording. Tyrosine phosphorylation of immunoprecipitated Na(V)1.5 was increased in cells expressing Fyn(CA) compared with Fyn(KD). We show that Fyn is present in rat cardiac myocytes, and that Na(V)1.5 channels from these myocytes are tyrosine-phosphorylated. In HEK-293 cells the effect of Fyn(CA) on Na(V)1.5 inactivation is abolished by the single point mutation Y1495F, a residue located within the cytoplasmic linker between the third and fourth homologous domains of the sodium channel. We provide evidence that this linker is a substrate for Fyn in vitro, and that Y1495 is a preferred phosphorylation site. These results suggest that cardiac sodium channels are physiologically relevant targets of Src family tyrosine kinases.

A tctex1-Ca2+ channel complex for selective surface expression of Ca2+ channels in neurons.

Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.

A protein phosphatase 2calpha-Ca2+ channel complex for dephosphorylation of neuronal Ca2+ channels phosphorylated by protein kinase C.

Phosphorylation and dephosphorylation are primary means for rapid regulation of a variety of neuronal functions, such as membrane excitability, neurotransmitter release, and gene expression. Voltage-gated Ca2+ channels are targets for phosphorylation by a variety of second messengers through activation of different types of protein kinases (PKs). Protein phosphatases (PPs), like PKs, are equally important in regulating Ca2+ channels in neurons. However, much less is understood about whether and how a particular type of PP contributes to regulating neuronal Ca2+ channel activities. This is primarily because of the lack of specific inhibitors/activators for different types of PPs, particularly the PP2c family. The functional roles of PP2c and its substrates in the brain remain virtually unknown. During our yeast two-hybrid screening, PP2calpha was pulled out by both N- and P/Q-type Ca2+ channel C termini. This raised the possibility that PP2calpha might be associated with voltage-gated Ca2+ channels for regulation of the Ca(2+) channel activity. Biochemical studies show that PP2calpha binds directly to neuronal Ca2+ channels forming a functional protein complex in vivo. PP2calpha, unlike PP1, PP2a and PP2b, is more effective in dephosphorylation of neuronal Ca2+ channels after their phosphorylation by PKC. In hippocampal neurons, disruption of the PP2calpha-Ca2+ channel interaction significantly enhances the response of Ca2+ channels to modulation by PKC. Thus, the PP2calpha-Ca2+ channel complex is responsible for rapid dephosphorylation of Ca2+ channels and may contribute to regulation of synaptic transmission in neurons.

Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis.

A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.

A PKC epsilon-ENH-channel complex specifically modulates N-type Ca2+ channels.

Multiple protein kinase C (PKC) isozymes are present in neurons, where they regulate a variety of cellular functions. Due to the lack of specific PKC isozyme inhibitors, it remains unknown how PKC acts on its selective target(s) and achieves its specific actions. Here we show that a PKC binding protein, enigma homolog (ENH), interacts specifically with both PKCepsilon and N-type Ca2+ channels, forming a PKCepsilon-ENH-Ca2+ channel macromolecular complex. Coexpression of ENH facilitated modulation of N-type Ca2+ channel activity by PKC. Disruption of the complex reduced the potentiation of the channel activity by PKC in neurons. Thus, ENH, by interacting specifically with both PKCepsilon and the N-type Ca2+ channel, targets a specific PKC to its substrate to form a functional signaling complex, which is the molecular mechanism for the specificity and efficiency of PKC signaling.