Identification and validation of novel signature associated with hepatocellular carcinoma prognosis using Single-cell and WGCNA analysis.

Background: Hepatocellular carcinoma is a rapidly advancing malignancy with a poor prognosis. Therefore, further research is needed on its potential pathogenesis and therapeutic targets. Methods: In this study, the relevant datasets were downloaded from the TCGA database and the key modules were identified using WGCNA in the necroptosis-related gene set, while single-cell datasets were scored using the necroptosis gene set. Differential genes in the high- and low-expression groups were determined using the WGCNA module genes as intersection sets to identify key genes involved in necroptosis in liver cancer. Then, prognostic models were constructed using LASSO COX regression followed by multi-faceted validation. Finally, model genes were found to be correlated with key proteins of the necroptosis pathway and used to identify the most relevant genes, followed by their experimental validation. Subsequently, on the basis of the analysis results, the most relevant SFPQ was selected for cell-level verification. Results: We constructed a prognosis model that included five necroptosis-related genes (EHD1, RAC1, SFPQ, DAB2 and PABPC4) to predict the prognosis and survival of HCC patients. The results showed that the prognosis was more unfavorable in the high-risk group compared to the low-risk group, which was corroborated using ROC curves and risk factor plots. In addition, we further checked the differential genes using GO and KEGG analyses and found that they were predominantly enriched in the neuroactive ligand-receptor interaction pathway. The results of the GSVA analysis demonstrated that the high-risk group was mainly enriched in DNA replication, regulation of the mitotic cycle, and regulation of various cancer pathways, while the low-risk group was predominantly enriched in the metabolism of drugs and xenobiotics using cytochrome P450. SFPQ was found to be the main gene that affects the prognosis and SFPQ expression was positively correlated with the expression of RIPK1, RIPK3 and MLKL. Furthermore, the suppression of SFPQ could inhibit hyper-malignant phenotype HCC cells, while the WB results showed that inhibition of SFPQ expression also resulted in lower expression of necroptosis proteins, compared to the sh-NC group. Conclusions: Our prognostic model could accurately predict the prognosis of patients with HCC to further identify novel molecular candidates and interventions that can be used as alternative methods of treatment for HCC.

[Effects of Huangpu Tongqiao Capsules on EGFR-PLCγ signal pathway of hippocampus in rats with Alzheimer's disease].

Huangpu Tongqiao Capsules(HPTQC), with the functions of invigorating Qi and kidney, eliminating phlegm and removing blood stasis, have the effect of treating Alzheimer's disease(AD), but its mechanism needs further exploration. To explore the relationship between the therapeutic mechanism of HPTQC on Alzheimer's disease and EGFR-PLCγ signal pathway, 40 healthy male SD rats were selected and divided into 4 groups randomly: sham operation group(sham), model group(model), HPTQC group(HPTQC), and nimodipine group(NMP). AD rat model was established by intraperitoneal injection of D-galactose combined with an intracerebral injection of amyloid-ß peptide(25-35). After 28 days of administration, Morris water maze test and HE staining showed that the learning and memory ability of AD rats were significantly decreased(P<0.01), and hippocampal neurons were obviously da-maged. However, HPTQC could improve the learning and memory ability of AD rats(P<0.05) and reduce the damage of hippocampal neurons. Immunofluorescence test results showed that the expression levels of EGFR and p-Tau in hippocampal CA1 region of AD rats were significantly increased(P<0.01), and HPTQC could reduce the expression of EGFR and p-Tau in hippocampus of AD rats(P<0.01). Western blot results showed that the protein expression levels of EGFR, PLCγ, IP3 R and p-Tau in hippocampus of AD rats were significantly increased(P<0.01), and HPTQC could reduce the protein expression of EGFR, PLCγ, IP3 R and p-Tau in AD rats(P<0.05). RT-PCR results showed that the mRNA levels of EGFR, PLCγ, IP3 R and Tau in hippocampus of AD rats were significantly increased(P<0.01), and HPTQC could reduce the mRNA levels of EGFR, PLCγ, IP3 R and Tau in AD rats(P<0.05). The results indicate that HPTQC can improve the learning and memory ability of AD rats, and its mechanism of action may be related to regulating EGFR-PLCγ signal pathway.

Transcriptomic and proteomic response of Manihot esculenta to Tetranychus urticae infestation at different densities.

Tetranychus urticae (Acari: Tetranychidae) is an extremely serious cassava (Manihot esculenta) pest. Building a genomic resource to investigate the molecular mechanisms of cassava responses to T. urticae is vital for characterizing cassava resistance to mites. Based on the tolerance of cassava varieties to mite infestation (focusing on mite development rate, fecundity and physiology), cassava variety SC8 was selected to analyze transcriptomic and proteomic changes after 5 days of T. urticae feeding. Transcriptomic analysis revealed 698 and 2140 genes with significant expression changes under low and high mite infestation, respectively. More defense-related genes were found in the enrichment pathways at high mite density than at low density. In addition, iTRAQ-labeled proteomic analysis revealed 191 proteins with significant expression changes under low mite infestation. Differentially expressed genes and proteins were mainly found in the following defense-related pathways: flavonoid biosynthesis, phenylpropanoid biosynthesis, and glutathione metabolism under low-density mite feeding and plant hormone signal transduction and plant-pathogen interaction pathways under high-density mite feeding. The plant hormone signal transduction network, involving ethylene, jasmonic acid, and salicylic acid transduction pathways, was explored in relation to the M. esculenta response to T. urticae. Correlation analysis of the transcriptome and proteome generated a Pearson correlation coefficients of R = 0.2953 (P < 0.01), which might have been due to post-transcriptional or post-translational regulation resulting in many genes being inconsistently expressed at both the transcript and protein levels. In summary, the M. esculenta transcriptome and proteome changed in response to T. urticae, providing insight into the general activation of plant defense pathways in response to mite infestation.

Unravelling the phylogeny, cryptic diversity and morphological evolution of Diptilomiopus mites (Acari: Eriophyoidea).

The Eriophyoidea, notable for specific morphological characters (four-legged mites) and gall-formation in host plants (gall mites), is one of the most species-rich superfamilies of Acari. Monophyly of the superfamily Eriophyoidea is accepted by all acarologists; however, monophyly of most genera has not been evaluated in a molecular phylogenetic network. Furthermore, most eriophyoid mites, especially species in the genus Diptilomiopus, are morphologically similar, challenging their identification. Here we test the phylogeny and cryptic diversity of Diptilomiopus species using fragments of two mitochondrial (COI and 12S) and two nuclear (18S and 28S) genes. Our results revealed the monophyly of Diptilomiopus. Sequence distance, barcode gap, and species delimitation analyses of the COI gene allowed us to resolve cryptic diversity of Diptilomiopus species. Additionally, we supposed that characteristics of genu fused with femur on both legs and seta ft' absent on leg II evolved only once within Diptilomiopus, which are potential morphological synapomorphies. In contrast, characteristics of both setae ft' and ft″ divided into a short branch and a long branch were supposed evolving multiple times independently. Our findings contribute to the understanding of phylogeny and morphological evolution of Diptilomiopus species and provide a DNA-based approach for species delimitation of Diptilomiopus mites.

Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1ß, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1ß, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

Cytotoxic activity of the alkaloids from Broussonetia papyrifera fruits.

CONTEXT: Broussonetia papyrifera (L.) Vent. (Moraceae), a traditional Chinese medicinal herb, has been extensively applied for many years to treat various diseases. Recently, a number of compounds with biological and pharmacological activities have been extracted from the plant and used as chemotherapeutic candidates to treat a range of diseases such as cancer. OBJECTIVE: The current study was designed to isolate the alkaloid compounds from ethyl acetate extraction of Broussonetia papyrifera fruits, and to evaluate the cytotoxic activity of total alkaloids as well as individual isoquinoline alkaloids from B. papyrifera fruits. METHODS: Alkaloid compounds were isolated from the ethyl acetate extraction by silica gel column chromatography methods using CHCl3/MeOH as eluents. The compounds' structures were determined by detailed analysis of NMR, MS spectral data, and chemical methodology. Cytotoxic activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods against human A375, Hela, BEL-7402 cancer cells, and non-cancer cells. RESULTS: Two isoquinonline alkaloids were isolated and characterized as N-norchelerythrine and dihydrosanguinarine. The total alkaloids and seven individual alkaloids had higher activities on BEL-7402 and Hela cell lines with low IC50 values 6.61-47.41 and 5.97-40.17 µg/mL (<50 µg/mL). Nitidine, broussonpapyrine, and chelerythrine had strong toxic on non-cancer cells with IC50 value 18.01, 19.91, and 22.31 µg/mL, respectively. DISCUSSION: N-Norchelerythrine and dihydrosanguinarine were isolated from this plant for the first time. Our data implicated that seven isoquinoline alkaloids had cytotoxity with structure-activity relationships, which provided fundamental information for further modification of their anticancer effect.

Intestinal aGVHD and infection after hematopoietic stem cell transplantation.

BACKGROUND: We aimed to guide clinical nursing by studying the relationship between intestinal acute graft-versus-host disease and intestinal infection after hematopoietic stem cell transplantation. MATERIAL/METHODS: We present an effective nursing method by comparing and analyzing the degree, duration time, and volume of diarrhea, and the distribution of pathogens in 44 patients who developed intestinal aGVHD after hematopoietic stem cell transplantation (24 patients with no intestinal infection). RESULTS: 21.4% of patients with grade I-II intestinal aGVHD developed into intestinal infection and 87.5% of patients with grade III-IV intestinal aGVHD developed into intestinal infection (P<0.05). Higher mortality was found in the grade III-IV intestinal aGVHD patients with intestinal infection. Patient age had no effect on the incidence of GVHD according to our data (P<0.05). We found remarkable differences in the amount and duration of diarrhea between patients with and without intestinal infection (P<0.05). The most common pathogens cultivated were Candida glabrata (24%) and Candida albicans (22.67%). CONCLUSIONS: The incidence of intestinal infection increased remarkably after intestinal aGVHD occurred. Severe aGVHD can easily lead to fungus infection. Nursing care can decrease the incidence of intestinal infection in aGVHD.

Retrograde type A aortic dissection during or after thoracic endovascular aortic repair: a single center 16-year experience.

Objective: This article aims to investigate the incidence rate of retrograde type A aortic dissection (RTAD) and the risk factors of RTAD in relation to thoracic endovascular aortic repair (TEVAR). Methods: Patients with thoracic aortic disease who underwent TEVAR at Henan Provincial People's Hospital from January 2004 to December 2019 were enrolled in the present research. The risk factors associated with RTAD following TEVAR using univariate and multiple logistic regression analyses. Results: During the study period, A total of 1,688 TEVAR patients were included in this study, and of these, 1,592 cases were included in the type B aortic dissection (TBAD) group, and 96 cases were included in the non-TBAD group. There were 1,230 cases of aortic dissection and 362 cases of aortic intramural hematoma and/or penetrating ulcer in the TBAD group. The non-TBAD group included 68 cases of thoracic aortic aneurysm, 21 cases of thoracic aortic pseudoaneurysm, and seven cases of congenital aortic coarctation. The overall incidence rate of RTAD was 1.1% (18/1,688) in patients, all of which occurred in the TBAD group. The cohort comprised 18 RTAD patients with an average age of 56.78, consisting of 13 males and 5 females. Among them, 13 individuals exhibited hypertension. Ten instances happened within the TEVAR perioperative period, including two cases during the surgery, six cases occurred within three months, two cases occurred after one year, and the longest interval was 72 months following TEVAR. TEVAR was successfully implemented in 17 patients, while the operation technique was temporarily altered in one case. The new entry position for RTAD was identified as the proximal region of the stent graft (SG) in 13 patients, while in five cases, the entry site was more than 2 cm away from the proximal region of the SG. 17 cases were at the greater curvature of the aorta, and one case was at the lesser curvature. Multivariate logistic regression analysis revealed that the SG oversizing ratio is a relevant risk factor for RTAD. However, ascending aortic diameter, aortic arch type, SG type, and anchored region were not directly related to the occurrence of RTAD. Conclusion: RTAD is a rare yet catastrophic complication. It could occur both during the procedure, early and late postoperative periods. Maintaining an appropriate SG oversizing ratio is crucial to minimize the risk of RTAD.

A neural circuit for gastric motility disorders driven by gastric dilation in mice.

Introduction: Symptoms of gastric motility disorders are common clinical manifestations of functional gastrointestinal disorders (FGIDs), and are triggered and exacerbated by stress, but the neural pathways underpinning them remain unclear. Methods: We set-up a mouse model by gastric dilation (GD) in which the gastric dynamics were assessed by installing strain gauges on the surface of the stomach. The neural pathway associated with gastric motility disorders was investigated by behavioral tests, electrophysiology, neural circuit tracing, and optogenetics and chemogenetics involving projections of the corticotropin-releasing hormone (CRH) from the paraventricular nucleus of the hypothalamus (PVN) to acetylcholine (ChAT) neurons in the dorsal motor nucleus of the vagus (DMV). Results: We found that GD induced gastric motility disorders were accompanied by activation of PVN CRH neurons, which could be alleviated by strategies that inhibits the activity of PVN CRH neurons. In addition, we identified a neural pathway in which PVN CRH neurons project into DMV ChAT neurons, modulated activity of the PVN CRH →DMV ChAT pathway to alleviate gastric motility disorders induced by GD. Discussion: These findings indicate that the PVN CRH →DMV ChAT pathway may mediate at least some aspects of GD related gastric motility, and provide new insights into the mechanisms by which somatic stimulation modulates the physiological functions of internal organs and systems.

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating ß-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and ß-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating ß-hemoglobinopathies.

Two new species and a new record of eriophyoid mites (Acari: Eriophyodea) from Guangxi, China.

Two new species of eriophyoid mites are described and illustrated from Guangxi, China: Acaphylla quercus sp. nov. collected on Quercus glauca Thunb. (Fagaceae), Rhyncaphytoptus miliusius sp. nov. collected on Miliusa sinensis Finet Gagnep. (Annonaceae). We further report Cecidodectes euzonus Nalepa, 1917, collected on Trema tomentosa (Roxb.) H.Hara (Cannabaceae), for the first time from China. All the species are vagrants on lower leaf surface causing no apparent damage to their host plants.

[A case-control study on correlation of pesticide exposure with childhood acute leukemia].

OBJECTIVE: To evaluate the correlation of pesticide exposure with childhood acute leukemia. METHODS: An exploratory case-control study was conducted among childhood acute leukemia patients under 15 years old in Shanghai, China. From January 1st, 2006 to December 31st, 2008, a total of 80 newly diagnosed acute leukemia patients were recruited from Shanghai Children's Medical Center for the case group. Another 96 age-matched patients who visited the hospital for health examination, pediatric treatment or osteological therapy excluding hematological system diseases and neoplastic disease, were recruited for the control group. A questionnaire survey was conducted in both groups; and a 30 - 40 ml random urine sample was collected from each participant. Five types of organophosphorus pesticide metabolites was then detected among the samples, using Gas Chromatography with Flame Spectrophotometry. RESULTS: According to result of the questionnaire survey, more participants (55.0% (44/80)) in case group than in the control group (33.3% (32/96)) reported using mosquitocidal, which might increase the risk of childhood acute leukemia (OR = 2.444; 95%CI: 1.326 - 4.506). At the same time, the detection showed that the concentration (median) of organophosphate metabolites diethyl phosphate, dimethyl phosphate, dimethyl thiophosphate, diethyl thiophosphate and diethyl dithiophosphate in case group were 0.0682, 0.0082, 0.0183, 0.0233, 0.4259 µg/g Cr, which were all significantly higher than in control group (0.0865, 0.0025, 0.0112, 0.0123, 0.1207 µg/g Cr) except the concentration of diethyl phosphate (Z = -1.081, P = 0.279). The difference showed statistical significance (Z = -5.752, -2.800, -3.316, -8.120, P < 0.05). CONCLUSION: Pesticide exposure may be one of the risk factors for childhood acute leukemia.

[Effect of Sini San prescription on apoptosis and proliferation of hepatocellular carcinoma HepG2 cells].

Objective: To investigate the effects of Sini San prescription(SNS) on the proliferation and apoptosis of HepG2 cells and its molecular mechanism. Methods: The morphological changes of hepatocellular carcinoma HepG2 cells treated by SNS were observed by inverted microscope. MTT assay was used to detect the inhibitory effect of SNS on cell proliferation. Fluorescence staining and flow cytometry were employed to analyze the effect of SNS on apoptosis of HepG2 cells. Rho123 (Rhodamine 123) staining method was performed to detect the changes of mitochondrial membrane potential, and Western blot was used to evaluate the expression of proteins related to apoptosis. Results: The number of hepatocellular carcinoma HepG2 cells were significantly decreased (P＜0.01) and cells showed typical apoptotic cell morphology after treated with serum contained SNS. The inhibition rate of HepG2 cells was increased with the increase of concentration of serum contained SNS. The number of cells in G1 phase was significantly increased, while G2 phase was decreased after treated with serum contained SNS(P＜0.05).The apoptosis rate and mitochondrial membrane potential of HepG2 cells were significantly increased and decreased after treated with serum contained SNS(P＜0.05). The expression levels of Bax, caspase-3,-9 and cyt-c were significantly increased, while the expression of bcl-2 was decreased in HepG2 cells treated with serum contained SNS(P＜0.05).Conclusion: Sini San prescription can inhibit the proliferation of HepG2 cells and induce apoptosis by mitochondrial pathway.

[Studies on chemical constituents of fructus broussonetiae].

OBJECTIVE: To study the chemical constituents of Fructus Broussonetiae. METHODS: Column chromatography with silic gel was employed to isolate and purify the 80% alcohol extract of Fructus Broussonetia, and the constituents were identified by spectral methods. RESULTS: Six compounds were isolated from 80% ethanol extract. Their structures were identified as Isoterihanine (1), Chelerythrine (2), Trillin (3), Sucrose (4), beta-sitosterol (5) and Fucosterol (6). CONCLUSION: These compounds are isolated from Fructus Broussonetiae for the first time.

Semi-permanent smartphone adapter for microscopes: Design demonstration and workflow testing using a slit-lamp biomicroscope.

BACKGROUND: A smartphone microscope adapter is an alternative to a permanently mounted camera, which often requires a specially designed light path as well as a computer system for data storage and network connectivity. A common disadvantage of these adapters is that they need to be repeatedly mounted and dismounted; this is inconvenient during serial observations. PURPOSE: The study purpose is to develop and test a semi-permanent microscope adapter. METHODS: Our prototype was designed using three-dimensional (3D) computer-aided design software and was 3D printed for testing. Two tests were designed to quantify the workflow improvement by measuring the time required to complete specific movements, which simulated the actions of capturing photographs by using an adapter and a slit-lamp biomicroscope. Our prototype was the experimental adapter, and two commercially available products were used as references. A single-shot test measured the average time required to complete a photographing cycle, in which a single photograph was recorded. A multiple-shot test measured the time required to complete cycles in which a different number of photographs were captured; thus, the time required specifically for the preparation phase of photographing was determined. Both tests were performed by 4 researchers. RESULTS: The average time required to complete a cycle using our prototype in single-shot test was significantly shorter than that using one of the references. For the other reference, significances were shown in 2 testers' results. In the multiple-shot test, the linear regression analysis also showed that the preparation phase of our prototype was significantly shorter than the preparation phase of the two commercially available products (P < 0.001). CONCLUSIONS: The semi-permanent design may improve the workflow of photography using smartphone adapters, thereby reducing the time and effort required for preparing devices, particularly when making multiple consecutive observations or when the targets disappear fast.

Tracking pollutants in dietary fish oil: From ocean to table.

Dietary fish oil used in aquafeed transfers marine pollutants to farmed fish. However, the entire transfer route of marine pollutants in dietary fish oil from ocean to table fish has not been tracked quantitatively. To track the entire transfer route of marine pollutants from wild fish to farmed fish through dietary fish oil and evaluate the related human health risks, we obtained crude and refined fish oils originating from the same batch of wild ocean anchovy and prepared fish oil-containing purified aquafeeds to feed omnivorous lean Nile tilapia and carnivorous fatty yellow catfish for eight weeks. The potential human health risk of consumption of these fish was evaluated. Marine persistent organic pollutants (POPs) were concentrated in fish oil, but were largely removed by the refining process, particularly dioxins and polychlorinated biphenyls (PCBs). The differences in the POP concentrations between crude and refined fish oils were retained in the fillets of the farmed fish. Fillets fat content and fish growth were positively and negatively correlated to the final POPs deposition in fillets, respectively. The retention rates of marine POPs in the final fillets through fish oil-contained aquafeeds were 1.3%-5.2%, and were correlated with the POPs concentrations in feeds and fillets, feed utilization and carcass ratios. The dietary crude fish oil-contained aquafeeds are a higher hazard ratio to consumers. Prohibiting the use of crude fish oil in aquafeed and improving growth and feed efficiency in farmed fish are promising strategies to reduce health risks originating from marine POPs.

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.

[Cytoxic activities of total alkaloids isolated from fructus broussonetiae in vitro].

OBJECTIVE: To observe the cytoxic activities of total alkaloids isolated from Fructus Broussonetiae on the growth inhibition in five carcinoma cell lines. METHODS: The growth inhibition was analyzed by MTT, Cell colony in the Hela, BEL-7402, A375, SMM1990, Saos-2 cell lines. RESULTS: The growth of all tumor cells were inhibited by total alkaloids isolated from Fructus Broussonetiae. CONCLUSION: The results show that total alkaloids isolated from Fructus Broussonetiae possess the role of antitumors in cell culture.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA3, MeJA and Ag+, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Chlorella vulgaris Induces Apoptosis of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells.

OBJECTIVE: Chlorella vulgaris (C. vulgaris), a unicellular green microalga, has been widely used as a food supplement and reported to have antioxidant and anticancer properties. The current study was designed to assess the cytotoxic, apoptotic, and DNA-damaging effects of C. vulgaris growth factor (CGF), hot water C. vulgaris extracts, inlung tumor A549 and NCI-H460 cell lines. METHODS: A549 cells, NCI-H460 cells, and normal human fibroblasts were treated with CGF at various concentrations (0-300 µg/ml) for 24 hr. The comet assay and γH2AX assay showed DNA damage in A549 and NCI-H460 cells upon CGF exposure. Evaluation of apoptosis by the TUNEL assay and DNA fragmentation analysis by agarose gel electrophoresis showed that CGF induced apoptosis in A549 and NCI-H460 cells. RESULTS: Chlorella vulgaris hot water extract induced apoptosis and DNA damage in human lung carcinoma cells. CONCLUSION: CGF can thus be considered a potential cytotoxic or genotoxic drug for treatment of lung carcinoma.