Transcriptome analysis reveals key transcription factors and pathways of polian vesicle associated with cell proliferation in Vibrio splendidus-challenged Apostichopus japonicus.

Polian vesicle is thought to produce coelomocytes and contribute to the sea cucumber's immune system. Our previous work has indicated that polian vesicle was responsible for cell proliferation at 72 h post pathogenic challenge. However, the transcription factors related to the activation of effector factors and the molecular process behind this remained unknown. In this study, to reveal the early functions of polian vesicle in response to the microbe, a comparative transcriptome sequencing of polian vesicle in V. splendidus-challenged Apostichopus japonicus, including normal group (PV 0 h), pathogen challenging for 6 h (PV 6 h) and 12 h (PV 12 h) was performed. Compared PV 0 h to PV 6 h, PV 0 h to PV 12 h, and PV 6 h to PV 12 h, we found 69, 211, and 175 differentially expressed genes (DEGs), respectively. KEGG enrichment analysis revealed the DEGs, including several transcription factors such as fos, FOS-FOX, ATF2, egr1, KLF2, and Notch3 between PV 6 h and PV 12 h were consistently enriched in MAPK, Apelin and Notch3 signaling pathways related to cell proliferation compared with that in PV 0 h. Important DEGs involved in cell growth were chosen, and their expression patterns were almost the same as the transcriptome profile analysis by qPCR. Protein interaction network analysis indicated that two DEGs of fos and egr1 were probably significant as key candidate genes controlling cell proliferation and differentiation in polian vesicle after pathogenic infection in A. japonicus. Overall, our analysis demonstrates that polian vesicles may play an essential role in regulating proliferation via transcription factors-mediated signaling pathway in A. japonicus and provide new insights into hematopoietic modulation of polian vesicles in response to pathogen infection.

DNA-TCP complex structures reveal a unique recognition mechanism for TCP transcription factor families.

Plant-specific TCP transcription factors are key regulators of diverse plant functions. TCP transcription factors have long been annotated as basic helix-loop-helix (bHLH) transcription factors according to remote sequence homology without experimental validation, and their consensus DNA-binding sequences and protein-DNA recognition mechanisms have remained elusive. Here, we report the crystal structures of the class I TCP domain from AtTCP15 and the class II TCP domain from AtTCP10 in complex with different double-stranded DNA (dsDNA). The complex structures reveal that the TCP domain is a distinct DNA-binding motif and the homodimeric TCP domains adopt a unique three-site recognition mode, binding to dsDNA mainly through a central pair of ß-strands formed by the dimer interface and two basic flexible loops from each monomer. The consensus DNA-binding sequence for class I TCPs is a perfectly palindromic 11 bp (GTGGGNCCCAC), whereas that for class II TCPs is a near-palindromic 11 bp (GTGGTCCCCAC). The unique DNA binding mode allows the TCP domains to display broad specificity for a range of DNA sequences even shorter than 11 bp, adding further complexity to the regulatory network of plant TCP transcription factors.

Dimmed gene knockout shortens larval growth and reduces silk yield in the silkworm, Bombyx mori.

The bHLH domain transcription factor, Bombyx mori-derived dimmed (Bmdimm), is directly regulated by the JH-BmMet/BmSRC-BmKr-h1 pathway and plays a key role in regulating the expression of FibH, which codes the main component of silk protein. However, the other roles of Bmdimm in silk protein synthesis remain unclear. Here, we established a Bmdimm knockout (KO) line containing a 7-bp deletion via CRISPR/Cas9 system, which led to the absence of the bHLH domain. The expression level of silk protein genes and silk yield decreased significantly in the Bmdimm KO line. Moreover, knocking out Bmdimm led to shortened larval stages and significant weight loss in larvae and adults. Bmdimm was found to be highly expressed in the silk gland, but it was also expressed in the fat body. The expression level of Bmkr-h1 in the fat body was significantly downregulated in the Bmdimm KO line. Exogenous JHA treatment upregulated Bmkr-h1 and rescued the phenotype of larval growth in the Bmdimm KO line. In conclusion, knocking out Bmdimm led to a shortened larval stage via the inhibition of Bmkr-h1 expression, then reduced silk yield. These findings help to elucidate the regulatory mechanism of fibroin synthesis and larval development in silkworms.

Occurrence of antibiotics in waters, removal by microalgae-based systems, and their toxicological effects: A review.

Global antibiotics consumption has been on the rise, leading to increased antibiotics release into the environment, which threatens public health by selecting for antibiotic resistant bacteria and resistance genes, and may endanger the entire ecosystem by impairing primary production. Conventional bacteria-based treatment methods are only moderately effective in antibiotics removal, while abiotic approaches such as advanced oxidation and adsorption are costly and energy/chemical intensive, and may cause secondary pollution. Considered as a promising alternative, microalgae-based technology requires no extra chemical addition, and can realize tremendous CO2 mitigation accompanying growth related pollutants removal. Previous studies on microalgae-based antibiotics removal, however, focused more on the removal performances than on the removal mechanisms, and few studies have concerned the toxicity of antibiotics to microalgae during the treatment process. Yet understanding the removal mechanisms can be of great help for targeted microalgae-based antibiotics removal performances improvement. Moreover, most of the removal and toxicity studies were carried out using environment-irrelevant high concentrations of antibiotics, leading to reduced guidance for real-world situations. Integrating the two research fields can be helpful for both improving antibiotics removal and avoiding toxicological effects to primary producers by the residual pollutants. This study, therefore, aims to build a link connecting the occurrence of antibiotics in the aquatic environment, the removal of antibiotics by microalgae-based processes, and the toxicity of antibiotics to microalgae. Distribution of various categories of antibiotics in different water environments were summarized, together with the antibiotics removal mechanisms and performances in microalgae-based systems, and the toxicological mechanisms and toxicity of antibiotics to microalgae after either short-term or long-term exposure. Current research gaps and future prospects were also analyzed. The review could provide much valuable information to the related fields, and provoke interesting thoughts on integrating microalgae-based antibiotics removal research and toxicity research on the basis of environmentally relevant concentrations.

Comparison on nutritional quality of adult female Chinese mitten crab (Eriocheir sinensis) with different colored hepatopancreases.

White hepatopancreas syndrome has recently emerged in Chinese mitten crab (Eriocheir sinensis) aquaculture, causing considerable economic loss. The hepatopancreas color of diseased crabs becomes gradually lighter, turning from yellow to yellow-white to white. Therefore, this study was conducted to investigate the changes in nutrient composition in three edible parts (hepatopancreas, ovaries, and muscle) of adult females with different colored hepatopancreases. Three groups were assessed in this study, including a yellow hepatopancreas group (control, L * = 63.92, a * = 22.14, b * = 60.95), a yellow-white hepatopancreas group (YWHG, L * = 65.06, a * = 22.35, b * = 57.80), and a white hepatopancreas group (WHG, L * = 65.72, a * = 10.70, b * = 30.52). No statistically significant differences in average weight, tissue indices, and total edible yield were observed among the three crab groups (P >0.05). The moisture content of the hepatopancreases and ovaries in WHG was 56.12% and 9.23% higher than the control values (P <0.05), whereas hepatopancreas crude fat and ovary crude protein levels were 62.23% and 11.45% lower than the control values (P < 0.05). The total carbohydrate levels of the three edible tissues were significantly higher and the crude protein content of ovaries was significantly lower in YWHG (P < 0.05). Most amino acid levels in the WHG muscle and ovaries were significantly lower than the control (P < 0.05). Moreover, the hepatopancreas levels of total polyunsaturated fatty acids (PUFA) and n-6PUFA in WHG were 24.88% and 31.83% lower than in control group, whereas the hepatopancreas levels of total PUFA and n-6PUFA in YWHG were also 21.88% and 23.20% lower compared to the controls (P < 0.05). Overall, the growth and the edible parts were not affected in YWHG and WHG. Moreover, YWHG crabs exhibited few effects on nutritional value; however, the fatty acid composition of crabs was significantly changed. In contrast, WHG crabs exhibited poor nutritional quality. Nonetheless, the consumption of crabs with yellow-white or white hepatopancreases is not recommended since the animal also referred to as diseased crabs.

Dilation of the cystic duct confluence in laparoscopic common bile duct exploration and stone extraction in patients with secondary choledocholithiasis.

BACKGROUND: Many options exist for the management of cholelithiasis and secondary choledocholithiasis. Among them, laparoscopic common bile duct exploration (LCBDE) with choledocotomy followed by laparoscopic cholecystectomy has gained popularity. However, efforts should be made to ensure minimally invasive or noninvasive management of the common bile duct (CBD). The purpose of this study was to explore the clinical experience of non-invasive surgical modality, i.e., laparoscopic transcystic dilation of the cystic duct confluence in CBD exploration (LTD-CBDE), including feasibility, safety, adverse events, and incidence. METHODS: In this retrospective analysis, 68 patients were offered the LTD-CBDE technique from December 2015 to April 2018 based on patient's own intention. During the surgery, the cystic duct confluence was dilated with separation forceps and/or a columnar dilation balloon. Subsequently, CBD exploration and stone extraction were performed with a choledochoscope. The entrance of the CBD was covered with a cystic duct stump wall and was subjected to primary closure at the end of surgery. RESULTS: Forty-nine females and 19 males with cholelithiasis and secondary choledocholithiasis were included. The mean age was 53 years old (18 to 72 year). Of these patients, 62 (91.2%) were successfully treated with the LTD-CBDE technique, and bile leakage was observed in 3 patients (4.4%). The mean operation time was 106 min, and the mean hospital stay was 5.9 days. Among the other 6 patients, 3 were converted to open cholecystectomy due to severe fibrosis, unclear anatomical structure at Calot's triangle (n = 2) or Mirizze syndrome (n = 1); LCBDE was performed in 3 patients due to cystic duct atresia (n = 2) and low level of flow from the gallbladder duct into the CBD (n = 1). These patients had a smooth postoperative course. In total, 43/68 of the patients presented no radiological evidence of retained CBD stones at the postoperative follow-up (40 patients treated with LTD-CBDE) 1 year later. CONCLUSIONS: The current work suggests that LTD-CBDE for the management of cholelithiasis and secondary choledocholithiasis is a feasible, safe and effective technique with a low complication rate. LTD-CBDE offers another alternative for surgeons to treat patients in similar scenarios. However, additional randomized, controlled studies are needed to demonstrate its efficacy, safety, and impact on CBD stenosis.

Diversity and Dynamics of Yeasts During Vidal Blanc Icewine Fermentation: A Strategy of the Combination of Culture-Dependent and High-Throughput Sequencing Approaches.

In this study, attention has been focused on the ecology of yeasts during the spontaneous and inoculated fermentation processes of Vidal blanc icewine in northeast China, which is very important for screening autochthonous yeast strains, understanding the roles of these strains, and managing fermentation. The strategies were to conduct spontaneous and inoculated laboratory-scale fermentation processes simultaneously and to analyze the samples taken at different fermentation stages by culture-dependent and -independent methods. Three hundred and thirty-eight yeast strains were isolated and twelve genera were identified by sequencing. During the spontaneous fermentation process, non-Saccharomyces yeasts were predominant in the initial and middle stages, whereas Saccharomyces dominated in the later stages; Candida was preponderant in the whole process, and its abundance in the final stages was only lower than Saccharomyces. The inoculated fermentation was characterized by a predominance of Saccharomyces throughout the fermentation process; non-Saccharomyces yeasts were observed in the early stage. The internal transcribed spacer (ITS) 2 region gene was firstly used to analyze the yeast diversity in the samples during the icewine fermentation processes by high-throughput sequencing (HTS), and a more complex mycobiota was revealed. Moreover, the dynamics of other major fungi (mainly Davidiella and Alternaria) during icewine fermentation processes were also revealed, which have never been reported in icewine before.

Crystal Structure of Tetrameric Arabidopsis MYC2 Reveals the Mechanism of Enhanced Interaction with DNA.

Jasmonates (JAs) are essential plant hormones that play important roles in the regulation of plant growth and the response to environmental stress. In the JA signaling pathway, the core transcription factors are a class of basic helix-loop-helix (bHLH) proteins, including MYC2, MYC3, and MYC4, that have different regulatory capacities. Here, we report the 2.7 Å crystal structure of the MYC2 bHLH domain complexed with G-box DNA, showing a cis-tetrameric structure. Biochemical assays confirmed that full-length MYC2 forms a stable homo-tetramer both in solution and in DNA-bound states, whereas MYC3 forms only a homodimer. Isothermal titration calorimetry (ITC) assays demonstrated that tetramerization enhanced DNA binding affinity, and fluorescence resonance energy transfer (FRET) assay indicated DNA looping potential of tetrameric MYC2. Luciferase assay further confirmed the importance of tetramerization in transcriptional regulation. Our studies provide a mechanistic explanation for the regulatory differences of MYC transcription factors.

Upregulation of MiR-29b contributes to mesenchymal stem cell dysfunction in patients with severe pre-eclampsia.

The aim of this study was to investigate the role of miR-29b in the regulation of decidua-derived mesenchymal stem cells (dMSCs) to influence the pathogenesis of pre-eclampsia (PE). dMSCs were isolated from decidua tissue and characterized. The expression of miRNAs was evaluated by quantitative PCR. Overexpression and inhibition of miR-29b were achieved in dMSCs, and the effect of miR-29b overexpression on the migration and angiogenic potential of human umbilical vein endothelial cells (HUVEC) was assessed. Furthermore, we performed bioinformatic analyses, luciferase reporter gene assays, and gene expression analyses to identify the potential targets of miR-29b and to verify the effect of target genes on dMSC function. Upregulation of miR-29b was confirmed in severe PE patients. Overexpression of miR-29b attenuated cell proliferation of dMSCs. Overexpression of miR-29b in dMSCs decreased the migratory and tubule formation ability of HUVECs. Histone deacetylase 4 (HDAC4) was identified as a target gene of miR-29b. Decreased migration and tubule formation in HUVECs was observed upon incubation with conditioned medium from dMSCs treated with the HDAC inhibitor trichostatin A. Our results demonstrated that upregulation of miR-29b in dMSCs has an important paracrine effect and might be involved in the development of PE.

Implantable neurotechnologies: a review of integrated circuit neural amplifiers.

Neural signal recording is critical in modern day neuroscience research and emerging neural prosthesis programs. Neural recording requires the use of precise, low-noise amplifier systems to acquire and condition the weak neural signals that are transduced through electrode interfaces. Neural amplifiers and amplifier-based systems are available commercially or can be designed in-house and fabricated using integrated circuit (IC) technologies, resulting in very large-scale integration or application-specific integrated circuit solutions. IC-based neural amplifiers are now used to acquire untethered/portable neural recordings, as they meet the requirements of a miniaturized form factor, light weight and low power consumption. Furthermore, such miniaturized and low-power IC neural amplifiers are now being used in emerging implantable neural prosthesis technologies. This review focuses on neural amplifier-based devices and is presented in two interrelated parts. First, neural signal recording is reviewed, and practical challenges are highlighted. Current amplifier designs with increased functionality and performance and without penalties in chip size and power are featured. Second, applications of IC-based neural amplifiers in basic science experiments (e.g., cortical studies using animal models), neural prostheses (e.g., brain/nerve machine interfaces) and treatment of neuronal diseases (e.g., DBS for treatment of epilepsy) are highlighted. The review concludes with future outlooks of this technology and important challenges with regard to neural signal amplification.

An Integrated Wireless Power Management and Data Telemetry IC for High-Compliance-Voltage Electrical Stimulation Applications.

This paper describes a 13.56-MHz wireless power recovery system with bidirectional data link for high-compliance-voltage neural/muscle stimulator. The power recovery circuit includes a 2-stage rectifier, 2 LDOs and a high voltage charge pump to provide 3 DC outputs: 1.8 V, 3.3 V and 20 V for the stimulator. A 2-stage time division based rectifier is proposed to provide 3 DC outputs simultaneously. It improves the power efficiency without introducing any impact on the forward data recovery. The 20 V output is generated by a modified low ripple charge pump that reduces the ripple voltage by 40%. The power management system shows 49% peak power efficiency. The data link includes a clock and data recovery (CDR) circuit and a load shift keying (LSK) modulator for bidirectional data telemetry. The forward and backward data rates of the data telemetry are 61.5 kbps and 33.3 kbps, respectively. In addition, a power monitor circuit for closed-loop power control is implemented. The whole system has been fabricated in a 24 V HV LDMOS option 1.8 µ m CMOS process, occupying a core area of around 3.5 mm (2).

A WBAN based cableless ECG acquisition system.

A Wireless Body Area Network (WBAN) based 3-lead cableless electrocardiography (ECG) acquisition system is described. To enable truly cableless ECG monitoring, a new ECG measurement configuration and method that acquires ECG signals at individual lead locations referenced to a localized ground is proposed. The synthesized ECG signals are evaluated against the standard wired 3-lead configuration on the same test subject. Average Pearson correlation coefficients of 0.82, 0.95 and 0.86 have been achieved for Lead I, II and III signals respectively, demonstrating a high degree of similarity in the synthesized signals. Measurements are obtained via a custom wireless network platform utilizing a TDMA-based MAC protocol supporting the star topology and a proprietary front-end ECG acquisition system.

A compact, low input capacitance neural recording amplifier.

Conventional capacitively coupled neural recording amplifiers often present a large input load capacitance to the neural signal source and hence take up large circuit area. They suffer due to the unavoidable trade-off between the input capacitance and chip area versus the amplifier gain. In this work, this trade-off is relaxed by replacing the single feedback capacitor with a clamped T-capacitor network. With this simple modification, the proposed amplifier can achieve the same mid-band gain with less input capacitance, resulting in a higher input impedance and a smaller silicon area. Prototype neural recording amplifiers based on this proposal were fabricated in 0.35 µm CMOS, and their performance is reported. The amplifiers occupy smaller area and have lower input loading capacitance compared to conventional neural amplifiers. One of the proposed amplifiers occupies merely 0.056 mm(2). It achieves 38.1-dB mid-band gain with 1.6 pF input capacitance, and hence has an effective feedback capacitance of 20 fF. Consuming 6 µW, it has an input referred noise of 13.3 µVrms over 8.5 kHz bandwidth and NEF of 7.87. In-vivo recordings from animal experiments are also demonstrated.

[Determination of recombinant hirudin in urine of rat by spectrophotometric method].

To develop a spectrophotometric method for determining the concentration of recombinant hirudin (rH) in urine of rats. rH concentration was determined based on the rH inhibility to thrombin which hydrolyzed the Chromozym TH TH chromogenic substrate to form the specific pNA absorbed at 405 nm. The standard rH in rat urine was determined by the spectrophotometric method at concentration of 6.25 to 75 ng x mL(-1) with day and intra-day RSD < 10%, method recoveries of > 95% and the dilution recoveries of > 93%. The rH samples of rat urines which iv dose of 0.5, 1.0, and 2.0 mg x kg(-1) were collected and analyzed by the CSA method. Their cumulative excretion rH at 0-12 hr were (116.850 +/- 57.160), (235.544 +/- 39.375) and (474.986 +/- 85.426) microg x kg(-1). The calculated cumulative excretion rate of three doses is about 23% which indicates that the rH was eliminated in the way of a linear kinetics in rats. The rH content in rat urine could be measured by the spectrophotometric method accurately, reliably and sensitively for the rH urinary excretion dynamics study.

Pharmacokinetics study of recombinant hirudin in the plasma of rats using chromogenic substrate, ELISA, and radioisotope assays.

AIM: To compare the analytical methods used to study the pharmacokinetics of recombinant hirudin in the plasma of rats that had been injected with (125)I-recombinant hirudin. METHODS: 2.0 mg/kg (125)I-recombinant hirudin were injected intravenously into rats. The recombinant hirudins in the plasma was analyzed by chromogenic substrate assay, enzyme-linked immunosorbent assay (ELISA), total radioisotope assay (RA) and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA). RESULTS: The chromogenic substrate assay standard curve was linear over the concentration range from 3.12 to 40.00 ng/ml for the recombinant hirudin in plasma. The relative standard deviations (RSD) for the intra- and inter-day variation were 5.0 to 6.3% and 11.9 to 12.6%, respectively. The recoveries of recombinant hirudin was 89.8% to 100.7%. The limit of quantification (LOQ) was 3.12 ng/ml. The concentration-time curve of the recombinant hirudin in the plasma could be explained as a two-compartment model. Pharmacokinetic parameters, including the half-life of distribution phase (t1/2 α), the half-life of elimination phase (t1/2 ß), volume of apparent distribution (Vd), and area under the concentration-time curve from zero to infinite time (AUC0-t) were 7.59 min, 46.99 min, 0.17 L/kg, and 204.5 mg/L/min, respectively, as determined by chromogenic substrate assay; 6.41 min, 47.28 min, 1.24 L/kg, and 575.18 mg/L/min, respectively, as determined by ELISA; 3.69 min, 701.90 min, 0.04 L/kg, and 4189 mg/L/min, respectively as determined by RA; and 4.57 min, 724.9 min, 0.09 L/kg, and 2329 mg/L/min, respectively, as determined by TCA-RA. CONCLUSIONS: The chromogenic substrate assay on the concentration dynamics of the recombinant hirudin in the plasma is a specific, sensitive, and accurate analytical method for pharmacokinetic studies. Moreover, the pharmacokinetic parameters determined by the chromogenic substrate assay and ELISA are congruent except for AUC.

Neuroprotective properties of aucubin in diabetic rats and diabetic encephalopathy rats.

In this study, we determined the neuroprotective effect of aucubin on diabetes and diabetic encephalopathy. With the exception of the control group, all rats received intraperitoneal injections of streptozotocin (STZ; 60 mg/kg) to induce type 1 diabetes mellitus (DM). Aucubin (1, 5, 10 mg/kg ip) was used after induction of DM (immediately) and diabetic encephalopathy (65 days after the induction of diabetes). The diabetic encephalopathy treatment groups were divided into short-term and long-term treatment groups. Treatment responses to all parameters were examined (body weight, plasma glucose, Y-maze error rates and proportion of apoptotic cells). In diabetic rats, aucubin controlled blood glucose levels effectively, prevented complications, and improved the quality of life of diabetic rats. In diabetic encephalopathy, aucubin significantly rescued neurons in the hippocampal CA1 subfield and reduced working errors during behavioral testing. The significant neuroprotective effect of aucubin could be seen not only in the short term (15 days) but also in the long term (45 days), which was a highly encouraging finding. These data suggest that aucubin may be a potential neuroprotective agent.

Antitumor activity of lobaplatin alone or in combination with antitubulin agents in non-small-cell lung cancer.

OBJECTIVE: Lobaplatin is used to treat patients with breast cancer, small-cell lung cancer, and chronic myelogenous leukemia in China. In this study, we assessed the in-vitro and in-vivo activities of lobaplatin alone or in combination with antitubulin agents against human non-small-cell lung cancer (NSCLC). METHODS: The cytotoxicities of lobaplatin against NSCLC cells were determined by the sulforhodamine B (SRB) assay. Cell cycle analysis and apoptosis were assessed using flow cytometry, and the in-vivo antitumor activities were evaluated in human NSCLC xenografts in nude mice. RESULTS: The cytotoxicity of lobaplatin was similar to or higher than that of cisplatin and carboplatin, with a mean IC(50) of 2.5 µmol/l in a variety of NSCLC cells. In addition, lobaplatin arrested cells in the S phase and triggered apoptosis. The combination of lobaplatin with antitubulin agents yielded synergistic cytotoxic activity in vitro. In NSCLC xenografts, lobaplatin alone showed significant antitumor activity. The combination of lobaplatin with antitubulin agents, especially with paclitaxel, led to significantly enhanced activity, which was superior to that of cisplatin combined with antitubulin agents. CONCLUSION: These data demonstrate that the use of lobaplatin alone or in combination with antitubulin agents might be a rational and novel therapeutic strategy for human NSCLC and warrants further clinical investigation.

Identification of a new locoweed (Oxytropis serioopetala) and its clinical and pathological features in poisoned rabbits.

By a series of experiments, we identified a new member of the locoweed family, Oxytropis serioopetala, that produces swainsonine, a phytotoxin harmful to livestock. In order to evaluate the toxicity of Oxytropis serioopetala, its extract was administered to ten rabbits by gavage at a dose of 1.5 mg/kg body weight as swainsonine once daily. After the 20th day, the rabbits appeared depressive and anorexic. In addition, intention tremors were apparent upon movement. Their eyes were dull. The rear limbs were severely weak and even progressed to partial paresis. The activities of serum aspartate aminotransferase (AST), alanine transaminase (ALT) and alkaline phosphatase (AKP) and urea nitrogen (BUN) levels in the poisoned rabbits increased significantly. Serum α-mannosidase (AMA) activity decreased markedly. Pathomorphological lesions in the locoweed-poisoned rabbits developed severe microvacuolation of visceral and neurological tissue. Extensive vacuolation was observed in the liver, kidney and brain. These clinical and pathological features are similar to the symptoms of locoism.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists.

Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.