Chemogenetic Evolution of Diversified Photoenzymes for Enantioselective [2 + 2] Cycloadditions in Whole Cells.

Artificial photoenzymes with novel catalytic modes not found in nature are in high demand; yet, they also present significant challenges in the field of biocatalysis. In this study, a chemogenetic modification strategy is developed to facilitate the rapid diversification of photoenzymes. This strategy integrates site-specific chemical conjugation of various artificial photosensitizers into natural protein cavities and the iterative mutagenesis in cell lysates. Through rounds of directed evolution, prominent visible-light-activatable photoenzyme variants were developed, featuring a thioxanthone chromophore. They successfully enabled the enantioselective [2 + 2] photocycloaddition of 2-carboxamide indoles, a class of UV-sensitive substrates that are traditionally challenging for known photoenzymes. Furthermore, the versatility of this photoenzyme is demonstrated in enantioselective whole-cell photobiocatalysis, enabling the efficient synthesis of enantioenriched cyclobutane-fused indoline tetracycles. These findings significantly expand the photophysical properties of artificial photoenzymes, a critical factor in enhancing their potential for harnessing excited-state reactivity in stereoselective transformations.

SEpi-3D: soft epipolar 3D shape measurement with an event camera for multipath elimination.

Multipath in 3D imaging happens when one pixel receives light from multiple reflections, which causes errors in the measured point cloud. In this paper, we propose the soft epipolar 3D(SEpi-3D) method to eliminate multipath in temporal space with an event camera and a laser projector. Specifically, we align the projector and event camera row onto the same epipolar plane with stereo rectification; we capture event flow synchronized with the projector frame to construct a mapping relationship between event timestamp and projector pixel; we develop a multipath eliminating method that utilizes the temporal information from the event data together with the epipolar geometry. Experiments show that the RMSE decreases by 6.55mm on average in the tested multipath scenes, and the percentage of error points decreases by 7.04%.

Nomogram predicting long-term overall and cancer-specific survival of patients with buccal mucosa cancer.

BACKGROUND: Few models about the personalized prognosis evaluation of buccal mucosa cancer (BMC) patients were reported. We aimed to establish predictive models to forecast the prognosis of BMC patients. METHODS: The complete clinicopathological information of BMC patients from the surveillance, epidemiology and end results program was collected and reviewed retrospectively. Two nomograms were established and validated to predict long-term overall survival (OS) and cancer-specific survival (CSS) of BMC patients based on multivariate Cox regression survival analysis. RESULTS: 1155 patients were included. 693 and 462 patients were distributed into modeling and validation groups with 6:4 split-ratio via a random split-sample method. Based on the survival analysis, independent prognostic risk factors (variables that can be used to estimate disease recovery and relapse chance) influencing OS and CSS were obtained to establish nomograms. Then, we divided the modeling group into high- and low-risk cohorts. The low-risk cohort had improved OS and CSS compared to the high-risk cohort, which was statistically significant after the Log-rank test (p < 0.05). Furthermore, we used the concordance index (C-index), calibration curve to validate the nomograms, showing high accuracy. The decision curve analyses (DCA) revealed that the nomograms had evident clinical value. CONCLUSIONS: We constructed two credible nomogram models, which would give the surgeons reference to provide an individualized assessment of BMC patients.

Phosphorylation of keratin 18 serine 52 regulates mother-daughter centriole engagement and microtubule nucleation by cell cycle-dependent accumulation at the centriole.

Serine-52 (Ser52) is the major physiologic site of keratin 18 (K18) phosphorylation. Here, we report that serine-52 phosphorylated K18 (phospho-Ser52 K18) accumulated on centrosomes in a cell cycle-dependent manner. Moreover, we found that phospho-Ser52 K18 was located at the proximal end of the mother centriole. Transfection with the K18 Ser52 → Ala (K18 S52A) mutant prevented centriole localization of phospho-Ser52 K18 and resulted in separation of the mother-daughter centrioles. Inhibition of microtubule polymerization led to the disappearance of aggregated phospho-Ser52 K18 on the centrosome; removal of inhibitors resulted in reaccumulation of phospho-Ser52 K18 in microtubule-organizing centers. Transfection with a K18 S52A mutant inhibited microtubule nucleation. These results reveal a cell cycle-dependent change in centrosome localization of phospho-Ser52 k18 and strongly suggest that the phosphorylation status of Ser52 K18 of mother centrioles plays a critical role in maintaining a tight engagement between mother and daughter centrioles and also contributes to microtubule nucleation.

Palladium-Catalyzed C-H/C-H Cross-Coupling by Mechanochemistry: Direct Alkenylation and Heteroarylation of N1-Protected 1H-Indazoles.

C3-alkenylated and C3-(hetero)arylated 1H-indazoles are privileged structural motifs in numerous pharmaceuticals. Direct C3-alkenylation and C3-(hetero)arylation of 1H-indazoles have been significantly challenging because of the inert nature of this carbon center. Herein, we present an efficient mechanochemical strategy for palladium-catalyzed C-H/C-H cross-coupling to construct C3-alkenylated and C3-heteroarylated 1H-indazoles using low-cost copper oxidants with satisfactory product yields and broad functional group tolerance. The robustness of the developed protocols was further demonstrated by the unprecedented total mechanosynthesis of the intermediate of PLK4 inhibitor CFI-400945 and HIF-1α inhibitor YC-1.

Decarboxylative acylation of N-free indoles enabled by a catalytic amount of copper catalyst and liquid-assisted grinding.

A facile decarboxylative acylation of N-free indoles with α-ketonates via liquid-assisted grinding was reported. The reaction requires only a catalytic amount of Cu(OAc)2·H2O in combination with O2 as the terminal oxidant to give various 3-acylindoles with high efficiency. Additionally, this new methodology was applicable to a gram-scale synthesis.

Steroidal Constituents from Roots and Rhizomes of Smilacina japonica.

Four new steroidal constituents (1-4) along with two known steroidal glycosides (5 and 6) were isolated from the roots and rhizomes of Smilacina japonica. Analysis of their physicochemical properties and spectroscopic profiles identified the compounds as (25S)-5α-spirostan-9(11)-en-3ß, 17α-diol (1); (25S)-5α-spirostan-9(11)-en-3ß, 12ß-diol (2); (25S)-5α-spirostan-9(11)-en-3ß, 17α-diol-3-O-ß-d-glucopyranoside (3); (25S)-5α-spirostan-9(11)-en-3ß, 17α-diol-3-O-ß-d-glucopyranosyl-(1→2)-[ß-d-glucopyranosyl-(1→3)]-ß-d-galactopyranoside (4); japonicoside B (5); and japonicoside C (6). All six compounds showed cytotoxic activity against SMMC-7712, Bel-7402, A549, H460, and K562 human cancer cells.

Inhibition of growth and metastasis of triple-negative breast cancer targeted by Traditional Chinese Medicine Tubeimu in orthotopic mice models.

OBJECTIVE: Triple-negative breast cancer (TNBC) is highly invasive and metastatic, which is in urgent need of transformative therapeutics. Tubeimu (TBM), the rhizome of Bolbostemma paniculatum (Maxim.) Franquet, is one of the Chinese medicinal herbs used for breast diseases since the ancient times. The present study evaluated the efficacy, especially the anti-metastatic effects of the dichloromethane extract of Tubeimu (ETBM) on TNBC orthotopic mouse models and cell lines. METHODS: We applied real-time imaging on florescent orthotopic TNBC mice model and tested cell migration and invasion abilities with MDA-MB-231 cell line. Digital gene expression sequencing was performed and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis applied to explore the pathways influenced by ETBM. Moreover, quantitative real-time polymerase chain reactions (qRT-PCR) and Western blot were delivered to confirm the gene expression changes. RESULTS: ETBM exhibited noticeable control on tumor metastasis and growth of TNBC tumors with no obvious toxicity. In compliance with this, it also showed inhibition of cell migration and invasion in vitro. Its impact on the changed biological behavior in TNBC may be a result of decreased expression of integrin ß1 (ITGß1), integrin ß8 (ITGß8) and Rho GTPase activating protein 5 (ARHGAP5), which disabled the focal adhesion pathway and caused change in cell morphology. CONCLUSIONS: This study reveals that ETBM has anti-metastatic effects on MDA-MB-231-GFP tumor and may lead to a new therapeutic agent for the integrative treatment of highly invasive TNBC.

Bromide-assisted chemoselective Heck reaction of 3-bromoindazoles under high-speed ball-milling conditions: synthesis of axitinib.

A mechanically-activated chemoselective Heck coupling for the synthesis of 3-vinylindazoles has been developed with the aid of catalytic amounts of TBAB and NaBr as both dehalogenation restrainer and grinding auxiliary. After tuning of the chemical conditions and mechanical parameters, a series of non-activated 3-bromoindazoles and a broad scope of olefins worked well to give the corresponding coupling products in good to excellent yields. A further application of this protocol was performed in a two-step mechanochemical Heck/Migita cross coupling, which provided a highly efficient route for the synthesis of axitinib.

Norepinephrine inhibits mesenchymal stem cell chemotaxis migration by increasing stromal cell-derived factor-1 secretion by vascular endothelial cells via NE/abrd3/JNK pathway.

Mesenchymal stem cells (MSCs), which are physiologically maintained in vascular endothelial cell (VEC)-based niches, play a critical role in tissue regeneration. Our previous studies demonstrated that sympathetic denervation could promote MSC mobilization, thereby enhancing bone formation in distraction osteogenesis (DO), a self-tissue engineering for craniofacial and orthopeadic surgeries. However, the mechanisms on how sympathetic neurotransmitter norepinephrine (NE) regulates MSC migration are not well understood. Here we showed that deprivation of NE by transection of cervical sympathetic trunk (TCST) inhibited stromal cell-derived factor-1 (SDF-1) expression in the perivascular regions in rat mandibular DO. In vitro studies showed that NE treatment markedly upregulated p-JNK and therefore stimulated higher SDF-1 expression in VECs than control groups, and siRNA knockdown of the abrd3 gene abolished the NE-induced p-JNK activation. On the other hand, osteoblasts differentiated from MSCs showed an increase in SDF-1 secretion with lack of NE. Importantly, NE-treated VECs inhibited the MSC chemotaxis migration along the SDF-1 concentration gradient as demonstrated in a novel 3-chamber Transwell assay. Collectively, our study suggested that NE may increase the SDF-1 secretion by VECs via NE/abrd3/JNK pathway, thereby inhibiting the MSC chemotaxis migration from perivascular regions toward bone trabecular frontlines along the SDF-1 concentration gradient in bone regeneration.

Slug silencing inhibited perineural invasion through regulation of EMMPRIN expression in human salivary adenoid cystic carcinoma.

Salivary adenoid cystic carcinoma (SACC) is the most frequent salivary gland malignancy with a unique characteristic that has been named perineural invasion (PNI). EMMPRIN is a transmembrane glycoprotein that has been demonstrated to promote PNI in SACC. Slug, one of the most effective promoters of the epithelial-to-mesenchymal transition (EMT), has been found to be associated with PNI in SACC. The aim of the present study was to investigate the roles and relationships of Slug, EMMPRIN, and E-cadherin in the PNI process of SACC. The expression levels of Slug, EMMPRIN, and E-cadherin in 115 primary SACC cases were statistically analyzed by immunohistochemistry. Simultaneously, the SACC cell line SACC-83 was transfected with recombinant plasmids of silencing Slug (si-Slug) and/or silencing EMMPRIN (si-EMMPRIN). The functions of Slug and EMMPRIN in the EMT and PNI process were assessed by reverse transcription PCR (RT-PCR), western blotting, morphological observation, scratch test, migration assay, and in vitro perineural invasion assay. The immunohistochemical statistics revealed that the high expression of Slug and EMMPRIN and the low expression of E-cadherin were significantly associated with the PNI of SACC (P < 0.05). Slug expression was significantly associated with EMMPRIN expression (P < 0.05), and Slug expression and EMMPRIN expression were both significantly negatively associated with E-cadherin expression (P < 0.05). Slug and EMMPRIN silencing both significantly inhibited EMMPRIN expression but promoted E-cadherin expression in SACC-83 cells (P < 0.01). The series of in vitro assays revealed that silencing of Slug, EMMPRIN, or both induced cell morphology changes and inhibited tumor cell motility and PNI ability in SACC-83 cells (P < 0.01). These results suggested that Slug silencing could inhibit the EMT process by downregulating EMMPRIN and then upregulating E-cadherin in the PNI process of SACC. The present study indicated that Slug and EMMPRIN are potential biomarkers and therapeutic targets for the diagnosis and treatment of PNI in human SACC.

Targeting P38 Pathway Regulates Bony Formation via MSC Recruitment during Mandibular Distraction Osteogenesis in Rats.

Distraction osteogenesis (DO) is a widely used self-tissue engineering. However, complications and discomfort due to the long treatment period are still the bottleneck of DO. Novel strategies to accelerate bone formation in DO are still needed. P38 is capable of regulating the osteogenic differentiation of both mesenchymal stem cells (MSCs) and osteoblasts, which are crucial to bone regeneration. However, it is not clear whether targeting p38 could regulate bony formation in DO. The purpose of the current work was to investigate the effects of local application of either p38 agonist anisomycin or p38 inhibitor SB203580 in a rat model of DO. 30 adult rats were randomly divided into 3 groups: (A) rats injected with DMSO served as the control group; (B) rats injected with p38 agonist anisomycin; (C) rats injected with p38 inhibitor SB203580. All the rats were subjected to mandibular distraction and the injection was performed daily during this period. The distracted mandibles were harvested on days 15 and 30 after surgery and subjected to the following analysis. Micro-computed tomography and histological evaluation results showed that local application of p38 agonist anisomycin increased new bone formation in DO, whereas p38 inhibitor SB203580 decreased it. Immunohistochemical analysis suggested that anisomycin promoted MSC recruitment in the distraction gap. In conclusion, this study demonstrated that local application of p38 agonist anisomycin can increase new bone formation during DO. This study may lead to a novel cell-based strategy for the improvement of bone regeneration.

[Molecular cloning and characterization of a novel DoSWEET1 gene from Dendrobium officinale].

SWEET (sugars will be eventually exported transporters) constitute a large and conserved gene family of sugar transporters in eukaryotes, which are important in the cellular metabolisms, growth and development, and plant-microbe interaction in plants. In the present study, a full length cDNA of SWEET encoding gene, designed as DoSWEET1 (GenBank accession No. KT957550), was identified in Dendrobium officinale using RT-PCR and RACE approaches. DoSWEET1 was 1 150 bp in length and encoded a 262-aa protein with a molecular weight of 29.18 kD and an isoelectric point of 9.49. The deduced DoSWEET1 protein contained seven transmembrane regions and two conserved MtN3-slv domains (11-94, 130-212）. Multiple sequence alignment revealed that DoSWEET1 had high identities 45%-54.6%） with SWEET proteins from various plants. A neighbor joining phylogenetic analysis suggests that DoSWEET1 belonged to the class Ⅱ subgroup of the SWEET evolutionary tree, and was closely related to rice OsSWEET13, OsSWEET14, and OsSWEET15. qPCR analysis demonstrated that DoSWEET1 gene was differentially expressed in the three included organs of D. officinale, and the expression was most abundant in the roots at 9.88 fold over that of the stems, followed by that of the leaves with 2.85 fold higher. In the 3rd symbiotic germinating seeds infected by Tulasnella sp., the transcipts were dramatically induced by 1 359.06 fold over that in the ungerniamted control seeds, suggesting a vital role of the gene in the D. officinale symbiotic germination process. Molecular cloning and characterization of the novel DoSWEET1 gene provides a foundation for the functional study of the gene in sugar translocation during the D. officinale symbiotic germination process.

[Correlation analysis between effectiveness of element in rhizonsphere soil and quality of Danshen (Salvia miltiorrhiza)].

In order to investigate the content and distribution of available element in the rhizonsphere soil of the growing areas of Salvia miltiorrhiza Bunge, the contents of available element (N,P,K,B,Cu,Zn,Fe,Mn) in 26 soil samples were tested and evaluated. The results showed that the contents of available P and Fe were very plentiful, available K, Cu and Zn were rich, available N and Mn were deficient, available B was extremely deficient in all growing areas of S. miltiorrhiza of eight provinces in China. The correlation analysis showed that the contents of eight kinds of available elements were varying degree correlation. The stepwise regression analysis between the contents of available elements of rhizonsphere soil and ten kinds of active ingredients of Danshen (Salviae Miltiorrhizae Radix et Rhizoma) were researched. The results showed that the rates of contribution of available N,B,Mn and Fe to quality of Danshen were relatively large and they were the significant factors, and the other factors did not show statistical significance. The recommended fertilizing strategies is that the usage of N,B and Mn fertilizers should be controlled according to different stages of growth of S. miltiorrhiza, and P fertilizer should be reduced in all growing areas of S. miltiorrhiza.

ß2-Adrenoreceptor-Mediated Proliferation Inhibition of Embryonic Pluripotent Stem Cells.

Adrenoreceptors (ARs) are widely expressed and play essential roles throughout the body. Different subtype adrenoceptors elicit distinct effects on cell proliferation, but knowledge remains scarce about the subtype-specific effects of ß2-ARs on the proliferation of embryonic pluripotent stem (PS) cells that represent different characteristics of proliferation and cell cycle regulation with the somatic cells. Herein, we identified a ß2-AR/AC/cAMP/PKA signaling pathway in embryonic PS cells and found that the pathway stimulation inhibited proliferation and cell cycle progression involving modulating the stem cell growth and cycle regulatory machinery. Embryonic stem (ES) cells and embryonal carcinoma stem (ECS) cells expressed functional ß-ARs coupled to AC/cAMP/PKA signaling. Agonistic activation of ß-ARs led to embryonic PS cell cycle arrest and proliferation inhibition. Pharmacological and genetic analyzes using receptor subtype blocking and RNA interference approaches revealed that this effect selectively depended on ß2-AR signaling involving the regulation of AKT, ERK, Rb, and Cyclin E molecules. Better understanding of the effects of ß2-ARs on embryonic PS cell proliferation and cycle progression may provide new insights into stem cell biology and afford the opportunity for exploiting more selective ligands targeting the receptor subtype for the modulation of stem cells.

Elective neck dissection in patients with head and neck adenoid cystic carcinoma: an international collaborative study.

BACKGROUND: Adenoid cystic carcinoma (ACC) accounts for 3-5 % of all head and neck malignancies. Investigations of outcomes from elective neck dissection (END) for patients with ACC are sparse. This study aimed to assess the impact of END on the survival of patients with ACC. METHODS: This retrospective multicentered study investigated 270 patients who underwent neck dissection. A multivariate analysis assessed associations of clinical and histopathologic characteristics with survival outcomes. RESULTS: The primary tumor sites included the oral cavity in 250 patients (55 %), the major salivary glands in 133 patients (29 %), the sinonasal mucosa in 68 patients (15 %), and the larynx in six patients (1 %). The overall rate of occult nodal metastases among the patients who underwent END was 17 % (38/226). The highest incidence of occult nodal metastases was with the oral cavity (66 %). The 5-year overall survival (72 and 79 % for patients with or without END, respectively) and disease-specific survival (74 and 81 % for patients with or without END, respectively) were similar in the two groups. The subgroup analysis of patients according to the primary site showed no significant impact of END on outcome. In the multivariate analysis, primary site, T classification, and N classification were the only variables associated with outcome. CONCLUSIONS: The incidence of occult neck metastases among patients with ACC is 17 %. The highest incidence of occult metastases is with the oral cavity. Statistical analysis showed no survival advantage for patients who underwent END compared with those who did not.

Steroidal Saponins from the Roots and Rhizomes of Tupistra chinensis.

Two new furostanol saponins 1-2 and a new spirostanol saponin 3 were isolated together with two known furostanol saponins 4-5 from the roots and rhizomes of Tupistra chinensis. Their structures were characterized as 1ß,2ß,3ß,4ß,5ß,26-hexahydroxyfurost-20(22), 25(27)-dien-5,26-O-ß-d-glucopyranoside (1), 1ß,2ß,3ß,4ß,5ß,6ß,7α,23ξ,26-nona-hydroxyfurost- 20(22),25(27)-dien-26-O-ß-d-glucopyranoside (2), (20S,22R)-spirost-25 (27)-en-1ß,3ß,5ß- trihydroxy-1-O-ß-d-xyloside (3), tupisteroide B (4) and 5ß-furost-Δ25(27)-en-1ß,2ß,3ß,4ß,5ß,7α, 22ξ,26-octahydroxy-6-one-26-O-ß-d-glucopyranoside (5), respectively, by extensive use of spectroscopic techniques and chemical evidence. Additionally, the in vitro cytotoxic activity of 1-4 was evaluated on human A549 and H1299 tumor cell lines, and compound 3 exhibited cytotoxicity against A549 cells (IC50 86.63 ± 2.33 µmol·L-1) and H1299 cells (IC50 88.21 ± 1.34 µmol·L-1).

[Effect of licorice flavonoids on kainic acid-induced seizure in mice].

OBJECTIVE: To investigate the effect of licorice flavonoid (LF) on kainic acid (KA)-induced seizure in mice and its mechanism. METHODS: Male adult ICR mice were injected with 25 mg/kg KA to induce temporal lobe seizure. LF was administrated 7 d before seizure induction (pre-treatment) or 24 h after seizure induction (post-treatment) for 7 d. Acute seizure latency, seizure stage and duration were observed and compared between LF- and vehicle-treated mice. From d2 on, mice with status epilepticus were video-monitored for spontaneous seizures, 10 h/d for 6 w. Immunohistochemical analysis of BrdU and Timm staining was conducted to detect the neurogenesis and mossy fiber sprouting, respectively. RESULTS: No significant difference was observed in acute seizure latency, seizure stage and duration between LF-and vehicle-treated mice. KA-induced acute seizure resulted in spontaneous seizure in mice, and the seizure frequency was increased with time. Pre- and post-treatment with LF decreased seizure frequency from w3 after modeling [(0.58±0.15)/d, (0.38±0.38)/d vs (1.23±0.23)/d, P <0.05]. Furthermore, KA-induced seizure resulted in robust neurogenesis and mossy fiber sprouting, while treatment with LF both pre- and post- KA injection significantly inhibited neurogenesis (15.6±2.6, 17.1±3.1 vs 28.9±3.5, P <0.05) and mossy fiber sprouting (1.33±0.31, 1.56±0.42 vs 3.0±0.37, P <0.05). CONCLUSION: LF has no significant anti-seizure effect. However, it can decrease epileptogenesis through inhibition of neurogenesis and mossy fiber sprouting.

[Characteristics of inorganic elements in Panacis Majoris Rhizoma and relationship with its Daodi habitat].

OBJECTIVE: To investigate the Daodi habitat of Panacis Majoris Rhizoma by analyzing the characteristics of inorganic elements in Panacis Majoris Rhizoma from different habitats. METHODS: The contents of inorganic elements in Panacis Majoris Rhizoma from different habitats were determined by ICP-AES. The characteristics of inorganic elements in Panacis Majoris Rhizoma were analyzed by correlation analysis, principal component analysis and cluster analysis. RESULTS: It was showed that there was a correlation between the contents of inorganic elements and the medicine quality of Panacis Majoris Rhizoma; Fe, Cr, Al, Mg, Cd, Ca and Zn were principal components of Panacis Majoris Rhizoma; and the contents of inorganic elements in Panacis Majoris Rhizoma existed regional differences. CONCLUSION: The contents of inorganic elements Ca, Fe and Zn,especially the content of the essential trace elements Fe and Zn, can be used as one of the key reference for medicinal quality evaluation of Panacis Majoris Rhizoma; as well, Shaanxi Province is probably the Daodi habitat of Panacis Majoris Rhizoma.

[Influence of soil factors on quality of panacis majoris rhizoma].

OBJECTIVE: To research and screen the soil factors which influenced the quality of Panacis Majoris Rhizoma. METHODS: By determining the effective constituent of saponin of Panacis Majoris Rhizoma from different habitats, and the soil nutrient and heavy metal content, using correlation analysis, stepwise regression analysis and other statistical methods to analyze the soil factors on the quality of Panacis Majoris Rhizoma. RESULTS: The contents of ginsenoside Ro and chikusetsusaponin NVa of samples from different habitats were different, the corresponding soil nutrient and heavy metal content were also different. The content of ginsenoside-Ro was positively related with the soil pH value (P <0. 01) and negatively related with the soil available N(P < 0.05). The content of chikusetsusaponin IVa was positively related with the soil pH (P < 0.01) and copper content (P < 0.05), and negatively with the soil available N(P < 0.05) CONCLUSION: Soil factors are the leading factors that influence the quality of Panacis Majoris Rhizoma, soil pH and soil available N were the dominating factors.