GRK2 kinases in the primary cilium initiate SMOOTHENED-PKA signaling in the Hedgehog cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits.

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

[Changes in fingerprint and chromaticity values of Mori Cortex during stir-frying process].

This study aims to reveal the dynamics of the HPLC fingerprint, chromaticity values, and main chemical components of Mori Cortex during the stir-frying process. The fingerprints of raw and processed products of Mori Cortex were established. The content of mulberroside A, oxyresveratrol, kuwanon G, and kuwanon H in the samples and the chromaticity values of the samples were determined. Furthermore, the similarity evaluation of fingerprints and the correlation analysis between fingerprints and chromaticity values were carried out. The results showed that the fingerprints of raw and processed products of Mori Cortex had high similarity, and the overall changes in the content of the main chemical components in the stir-frying process were similar. According to the experience, when the stir-frying is moderate, the total chromaticity value difference |ΔE~*_(ab)| is above 1.5. With the extension of stir-frying time, the L~* and E~*_(ab) values keep decreasing, and the a~* value keeps increasing. The results of the correlation analysis between fingerprints and chromaticity values showed that peaks 1(5-hydroxy maltol), 2(mulberroside A), 3, 4, 6, 7, 11(oxyresveratrol), 14, 17(kuwanon G), and 18(kuwanon H) had significant correlations with the chromaticity values. Quantitative analysis of the four components with higher content showed that the content of the four components decreased to varying degrees when the stir-frying was excessive. In addition, 5-hydroxy maltol was produced after stir-frying of Mori Cortex, and the fingerprint and chromaticity values showed regular changes during the stir-frying process. The chromaticity can be included in the evaluation of the stir-frying process of Mori Cortex, which provides a reference for standardizing the quality of stir-fried Mori Cortex.

Diagnostic Value of Contrast-Enhanced Ultrasound Features for WHO/ISUP Grading in Renal Cell Carcinoma.

OBJECTIVES: By analyzing the differences of contrast-enhanced ultrasound (CEUS) features between low- and high-grade of WHO/ISUP grading, to explore the diagnostic value of CEUS in evaluating the prognosis of renal cell carcinoma (RCC). METHODS: The qualitative and quantitative features of CEUS in 69 patients with RCC confirmed by surgical pathology in the Lanzhou University Second Hospital from March to October 2021 were retrospectively analyzed. Patients were categorized into two groups: low-grade group (n = 22) and high-grade group (n = 47), with surgical pathology as reference standard. The diagnostic performance of statistically significant CEUS features was evaluated by receiver operating characteristic (ROC) curves. RESULTS: There were statistically significant differences in enhancement degree (P = .032) and quantitative features such as slopelesion (P = .034), the differences between lesion and cortex in arrive time (∆AT = ATlesion - ATcortex , P = .013), peak intensity(∆PI = [PIlesion - PIcortex ]/PIcortex , P = .003), area under the curve (∆Area = Arealesion - Areacortex , P = .008) in two groups, and the sensitivity was 70.2% and specificity was 71.4% of ∆PI, which has a high diagnostic performance in the differentiation of low-grade group from high-grade group (P = .005). CONCLUSIONS: CEUS features such as ∆PI, may help differentiate low-grade RCC from high-grade RCC. CEUS has a promising application prospect in preoperative evaluation of the prognosis of RCC.

[Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species].

In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 µL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.

Novel 4,5-dihydrospiro[benzo[c]azepine-1,1'-cyclohexan]-3(2H)-one derivatives as PARP-1 inhibitors: Design, synthesis and biological evaluation.

To further explore the research of novel PARP-1 inhibitors, we designed and synthesized a series of novel amide PARP-1 inhibitors based on our previous research. Most compounds displayed certain antitumor activities against four tumor cell lines (A549, HepG2, HCT-116, and MCF-7). Specifically, the candidate compound R8e possessed strong anti-proliferative potency toward A549 cells with the IC50 value of 2.01 µM. Compound R8e had low toxicity to lung cancer cell line. And the in vitro enzyme inhibitory activity of compound R8e was better than rucaparib. Molecular docking studies provided a rational binding model of compound R8e in complex with rucaparib. The following cell cycle and apoptosis assays revealed that compound R8e could arrest cell cycle in the S phase and induce cell apoptosis. Western blot analysis further showed that compound R8e could effectively inhibit the PAR's biosynthesis and was more effective than rucaparib. Overall, based on the biological activity evaluation, compound R8e could be a potential lead compound for further developing novel amide PARP-1 inhibitors.

A new homo-aro-cholestane glycoside from the rhizome of Paris polyphylla var. chinensis.

A new homo-aro-cholestane glycoside parispolyside H, along with nine known compounds, were isolated from 75% ethanolic extract of the rhizome of Paris polyphylla var. chinensis. Their chemical structures were elucidated on the basic of analysis of detailed spectroscopic and physicochemical properties. In addition, the isolated compounds (1, 6-9) were evaluated for their cytotoxic activity against HepG2 human liver cancer cell lines. Among them, four known compounds (6-9) showed cytotoxicity with IC50 values ranging from 0.41 to 3.6 µM.

The Attitude towards Preventive Measures and Knowledge of COVID-19 Inpatients with Severe Mental Illness in Economically Underdeveloped Areas of China.

Patients with severe mental illnesses (SMI) were at high risk of infection during Coronavirus Diseases 2019 (COVID-19) pandemic. This study examined hospitalized SMI patients' attitude and knowledge towards the COVID-19 infection. A cross-sectional survey was conducted in five psychiatric hospitals located in Gansu province, the most economically underdeveloped area in China. Patients' attitude towards preventive measures and knowledge of COVID-19 were measured by a self-report questionnaire. A total of 925 hospitalized patients with SMI were recruited. Of them, 84.8% (95%CI: 82.4%-87.1%) had positive attitudes towards preventive measures of the COVID-19 outbreak. Being married (OR: 1.55, 95%CI: 1.05-2.30) and a higher educational level (OR: 1.63, 95%CI: 1.12-2.38) were independently associated with positive attitudes towards COVID-19 preventive measures, whereas higher educational level was associated with better knowledge of the COVID-19 outbreak (ß: 0.231, P < 0.001). Patients mainly received COVID-19 relevant knowledge from public media (58.9%), followed by their clinicians (33.2%). Most hospitalized SMI patients in economically underdeveloped areas in China showed positive attitudes towards COVID-19 preventive measures. However, public health education on COVID-19 relevant knowledge by mental health professionals was inadequate to reduce the risk of transmission and infection.

[Complanatoside A and complanatoside B in rats administered with raw and salt-processed Astragali Complanati Semen: a pharmacokinetics study].

An UPLC-MS/MS method was developed to simultaneously determine complanatoside A and complanatoside B in rat plasma with rutin as the internal standard and applied to examine the effect of salt-processing on pharmacokinetics of these two flavonoid glycosides. The pharmacokinetic parameters were estimated using DAS 3.2.6 and subjected to independent sample t-test with SPSS 23.0. No significant difference in T_(max) of complanatoside B was observed between the raw and processed groups; however, in the processed group, C_(max) and AUC_(0-12 h) of complanatoside B increased obviously(P<0.05), while MRT_(0-12 h) decreased from(3.34±0.44) h to(1.81±0.36) h(P<0.05). C_(max) [(14.72±11.13) µg·L~(-1)] and MRT_(0-24) [(3.93±0.26) h] of complanatoside A in the raw group were statistically different from those [(35.64±21.99) µg·L~(-1),(1.43±0.24) h] in the processed group(P<0.05). As a result, salt-processing can facilitate the in vivo adsorption and accelerate the excretion of complanatoside A and complanatoside B.

High-dose chemotherapy followed by autologous stem cell transplantation for patients with refractory/relapsed classical Hodgkin lymphoma: a single center experience from China.

To evaluate the outcomes of refractory/relapsed cHL patients after high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT) in Beijing Cancer hospital and to identify the prognostic risk factors. We retrospectively analyzed 115 relapsed/refractory cHL patients who accepted HDCT and ASCT in our cancer center and had complete follow-up data from April 2000 to May 2017. Ages of these 115 patients at ASCT ranged from 14 to 63 (median age 28). Forty-four (38.3%) patients achieved CR and 50 (43.5%) patients achieved PR before ASCT. Thirty-seven (48.7%) patients of those 76 patients who did PET-CT before ASCT had negative PET-CT scans. The median follow-up time was 72 months. A total of 23 patients died in our study. The 5-year OS and PFS rates of all patients after ASCT were 78.7% and 53%, respectively. The 5-year OS rates after ASCT of patients who were in CR or PR or less than PR status before ASCT were 92.8%, 68.2%, and 76.2%, respectively (log-rank = 2.913, p = 0.233). And their 5-year PFS rates after ASCT were 69.2%, 54.2%, and 18.5%, respectively (log-rank = 13.615, p = 0.001). Univariate analysis revealed that ECOG (p = 0.010; hazard ratio = 1.578), disease status before ASCT (CR: p = 0.001; hazard ratio = 0.227) and after ASCT (CR: p < 0.001; hazard ratio = 0.154), and PET-CT results after ASCT (p = 0.023; hazard ratio = 0.438) significantly impact patients' PFS while number of pretransplant salvage chemotherapy (p = 0.037; hazard ratio = 2.521), radiotherapy (p = 0.046; hazard ratio = 0.423), and disease status after ASCT (CR: p = 0.010; hazard ratio = 0.197) significantly affected patients' OS. Multivariate analysis shown only disease status before ASCT (p = 0.002) had significant impact on PFS and disease status after ASCT (p = 0.021) had significant impact on OS. R/R HL patients can still obtain long-term PFS after HDCT and ASCT and disease status before ASCT was the most significant prognostic factor for PFS.

Design, synthesis and biological evaluation of homoerythrina alkaloid derivatives bearing a triazole moiety as PARP-1 inhibitors and as potential antitumor drugs.

A series of homoerythrina alkaloid derivatives containing a 1,2,3-triazole moiety as PARP-1 inhibitors were designed and synthesized. And their anti-proliferative activity was further evaluated. Compound 10n had excellent activity to inhibit proliferation of A549 cells (IC50 = 1.89 µM), which was higher than harringtonine (IC50 = 10.55 µM), pemetrexed (IC50 = 3.39 µM), and rucaparib (IC50 = 4.91 µM). Furthermore, the selectivity index of compound 10n was higher than rucaparib and pemetrexed for lung cancer cells. Flow cytometry analysis showed that compound 10n significantly arrested the cell cycle in the S phase, then induced apoptosis of A549 cells (apoptosis rate is 46%), which effectively inhibited cell proliferation. Simultaneously, western blot analysis revealed that compound 10n could prevent the biosynthesis of PAR. Further analysis results revealed that compound 10n could inhibit the expression of cyclin A, down-regulate the expression of bcl-2/bax, activate caspase-3, and ultimately induce apoptosis of A549 cells. All the results indicated that compound 10n had potential research value as a novel PARP-1 inhibitor in antitumor, and it provided a new reference for further development of PARP-1 inhibitors.

Design, synthesis and biological evaluation of erythrina derivatives bearing a 1,2,3-triazole moiety as PARP-1 inhibitors.

Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) have shown to be promising in clinical trials against cancer, and many researchers are interested in the development of new PARP-1 inhibitors. Herein, we designed and synthesized 44 novel erythrina derivatives bearing a 1,2,3-triazole moiety as PARP-1 inhibitors. MTT assay results indicated that compound 10b had the most potent anti-proliferative activity against A549 cells among five cancer cells. The enzyme inhibitory activity in vitro of compound 10b was also significantly better than rucaparib. Furthermore, the selectivity index of compound 10b was higher than rucaparib for lung cancer cells. Flow cytometry analysis showed that compound 10b induced apoptosis of A549 cells by the mitochondrial pathway. Western blot analysis indicated that compound 10b was able to inhibit the biosynthesis of PAR effectively, and it was more potent than rucaparib. Also, compound 10b was able to up-regulate the ratio of bax/bcl-2, activate caspase-3, and ultimately induced apoptosis of A549 cells. The combined results revealed that the discovery of novel non-amide based PARP-1 inhibitors have great research significance and provide a better choice for the future development of drugs.

Synthesis and biological evaluation of 7-methoxy-1-(3,4,5-trimethoxyphenyl)-4,5-dihydro-2H-benzo[e]indazoles as new colchicine site inhibitors.

The colchicine site inhibitors (CSIs) displayed both antimitotic and vascular disrupting activities, therefore are promising potential antitumor agents. In this study, a series 1-phenyl-4,5-dihydro-2H-benzo[e]indazoles were found as new CSIs of which the bioactive configuration was locked. Among them, compounds C1 and C2 displayed the best activity, with tubulin polymerization IC50 of 3.4 and 1.5 µM, and growth IC50 of low nanomolar concentrations against human colon cancer cell lines. In addition, compound C1 showed excellent broad-spectrum antitumor activity in the NCI-60 Human Tumor Cell Lines Screen, encouraging further study of this antitumor compound.

Design, synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target inhibitors for the treatment of multiple myeloma.

Multiple myeloma (MM) is the second most common haematological malignancy. Almost all patients with MM eventually relapse, and most recommended treatment protocols for the patients with relapsed refractory MM comprise a combination of drugs with different mechanisms of action. Therefore novel drugs are in urgent need in clinic. Bcl-2 inhibitors and HDAC inhibitors were proved their anti-MM effect in clinic or under clinical trials, and they were further discovered to have synergistic interactions. In this study, a series of Bcl-2/HDAC dual-target inhibitors were designed and synthesized. Among them, compounds 7e-7g showed good inhibitory activities against HDAC6 and high binding affinities to Bcl-2 protein simultaneously. They also displayed good growth inhibitory activities against human MM cell line RPMI-8226, which proved their potential value for the treatment of multiple myeloma.

Chemical constituents from the seeds of Cassia obtusefolia and their in vitro α-glucosidase inhibitory and antioxidant activities.

Basing on chromatographic separation techniques, fifteen aglycones (1-15), including two new anthraquinone aglycones (1, 2) and thirteen known compounds (3-15), were isolated from the small polar fraction of Cassia obtusefolia (petroleum ether extract). Structural elucidations were performed by 1D/2D NMR spectroscopy and mass spectrometry. The in vitro antioxidant and α-glucosidase inhibitory activities of these fifteen compounds were determined. Except compounds 12 (IC50 3.03 ±â€¯0.31 µg/mL, stronger than ascorbic acid, which IC50 was 6.48 ±â€¯2.30 µg/mL) and 13 (IC50 78.40 ±â€¯2.39 µg/mL), the free radical scavenging capacities of other compounds were weak. Compounds 4, 5, 6 and 13 exhibited inhibitory activities on α-glucosidase with IC50 values of 50.60 ±â€¯1.10, 22.57 ±â€¯0.07, 60.09 ±â€¯1.40, and 80.01 ±â€¯2.66 µg/mL separately, however, all the α-glucosidase inhibitory activities were weaker than positive control (acarbose).

[Effects of processing on pharmacokinetics of Descurainiae Semen's constituents in rats].

An UPLC-MS/MS method simultaneously determining contents of quercetin-3-O-ß-D-glucose-7-O-ß-D-gentiobioside and sinapic acid in rats' plasma was firstly established and applied to study the effects of processing on pharmacokinetics of Descurainiae Semen's active constituents. Complantatoside A as internal standard,methanol used for protein precipitation,the method was validated according to the instructions of CFDA. Rats' plasma was collected after being oral administrated equal dosage of 60% ethanal extract of raw or processed Descurainiae Semen at different point of time,then the concentrations were determined to calculate pharmacokinetic parameters using DAS 3. 2. 6. And the parameters were analyzed using SPSS 23. 0,meantime the concentration-time curve was drawn.The results showed that processing had no effects on the pharmacokinetics of QGG,but could improve the absorption of sinapic acid and slow down the excretion.

Impact of impaired glucose regulation on the functions of large and small fibers of peripheral nerves

Background/aim: This article analyzes the incidence and characteristics of peripheral neuropathy in patients with impaired glucose regulation (IGR). Materials and methods: A total of 120 IGR patients and 60 healthy controls were enrolled. All subjects underwent nerve conduction study (NCS) of large fibers and skin sympathetic response (SSR) and contact heat pain evoked potential (CHEP) testing of small fibers with a Medtronic Keypoint machine (Medoc Ltd., Israel). IGR patients were evaluated using the Michigan Neuropathy Screening Instrument (MNSI). Results: The abnormal rates (MNSI >2) in IGR patients and NCS and SSR evaluations were 18.3%, 22.5%, and 39.2%, respectively. All abnormal NCS findings were accompanied with abnormal SSR findings. Compared with the control group, the sensory nerve action potential wave of the posterior tibial and sural nerve was decreased in the IGR group (P = 0.01, P = 0.00), the SSR wave was reduced in the upper and lower limbs (P = 0.002, P = 0.00), and the CHEP wave was decreased in opisthenar and shank (P = 0.00). Compared with the control group, the CHEP wave was decreased in the shank in the normal SSR group (P < 0.05) and in the opisthenar and shank in the normal NCS group (P < 0.05). Conclusion: IGR patients have peripheral neuropathy characterized by impaired functions of large and small fibers focused on small fiber and lower limb sensory nerves. CHEP can detect small fiber damage earlier than SSR and NCS.

[Chemical study on Cassiae Semen processing procedure].

References and our previous experiment showed that the contents of glycosides were significantly decreased，while the contents of aglycones were significantly increased after processing of Cassiae Semen.It may be related to its glycosidases or the heating process. In order to investigate the reasons, high performance liquid chromatographic (HPLC) was used to study the effects of these two factors on contents of Cassiae Semen's main chemical components in processing. The results showed that glycoside hydrolases was present in Cassiae Semen and could rapidly hydrolyze glycosides from Cassiae Semen into aglycones in suitable temperature with sufficient water.However，it didn't show effect on contents change of main constituents in the procedure of Cassiae Semen processing.The reason for content decrease of glycosides and content increase of aglycones in processed Cassiae Semen was glycoside bond cracking to produce corresponding aglycone at high temperature.This study further provides basis for further revealing of the processing mechanism of Cassiae Semen.

[Chemical constituents from seeds of Astragali complanati].

A new flavonoid glycoside, named complanatoside C (1), and 19 known compounds (2-20) were isolated from an 95% ethanol extract of Astragali Semen by various chromatographic methods. Their structures were identified on the basis of UV, IR, NMR, MS spectroscopic data analysis, and comparison with those in literature, including fifteen flavonoid glycoside (1-15), and six other constituents (16-20), among which compounds 16-19 were isolated from this plant for the first time.