Causal association between major depressive disorder and venous thromboembolism: a bidirectional mendelian randomization study.

Purpose: Major depressive disorder (MDD) and venous thromboembolism (VTE) may be linked in observational studies. However, the causal association remains ambiguous. Therefore, this study investigates the causal associations between them. Methods: We performed a two-sample univariable and multivariable bidirectional Mendelian randomization (MR) analysis to evaluate the associations between MDD and VTE. The summary genetic associations of MDD statistics were obtained from the Psychiatric Genomics Consortium and UK Biobank. Information on VTE, deep vein thrombosis (DVT), and pulmonary embolism (PE) were obtained from the FinnGen Biobank. Inverse-variance weighting was used as the main analysis method. Other methods include weighted median, MR-Egger, Simple mode, and Weighted mode. Results: Univariable MR analysis revealed no significant associations between MDD and VTE risk (odds ratio (OR): 0.936, 95% confidence interval (CI): 0.736-1.190, p = 0.590); however, after adjusting the potential relevant polymorphisms of body mass index and education, the multivariable MR analysis showed suggestive evidence of association between them (OR: 1.163, 95% CI: 1.004-1.346, p = 0.044). Univariable MR analysis also revealed significant associations between MDD and PE risk (OR: 1.310, 95% CI: 1.073-1.598, p = 0.008), but the association between them was no longer significant in MVMR analysis (p = 0.072). We found no significant causal effects between MDD and DVT risk in univariable or multivariable MR analyses. There was also no clear evidence showing the causal effects between VTE, PE, or DVT and MDD risk. Conclusion: We provide suggestive genetic evidence to support the causal association between MDD and VTE risk. No causal associations were observed between VTE, PE, or DVT and MDD risk. Further validation of these associations and investigations of potential mechanisms are required.

Effects of dimpropyridaz on feeding behavior, locomotivity and biological parameters of Aphis gossypii.

Aphis gossypii is a worldwide agricultural pest insect that has developed resistance to multiple pesticides. Dimpropyridaz is a new chordotonal organ regulator and has been registered for control of sap-sucking insects including A. gossypii. For the aim to effectively apply dimpropyridaz for A. gossypii control, it is necessary to clarify the toxic effects of dimpropyridaz on cotton aphids. In the present study, the effects of dimpropyridaz on feeding behavior, locomotivity and biological parameters of A. gossypii were investigated. The bioassay results showed that dimpropyridaz had good insecticidal activity against A. gossypii, with LC50 as 1.91 mg/L at 72 h post exposure. Moreover, the dimpropyridaz treated A. gossypii showed obvious poisoning symptoms of dehydration and shrivel. Through the gentle-touch experiment and feeding experiment, it was found that dimpropyridaz treatment had significant adverse impacts on the locomotivity and feeding behavior of A. gossypii. Compared with the control group, the coordinated movement ability of the treated A. gossypii attenuated, moreover the feeding behavior of A. gossypii was inhibited. The feeding rate decreased by 62.00%, 64.00% and 71.67% after treatment with 50.33 mg/L dimpropyridaz for 24 h, 48 h and 72 h, respectively. Especially, EPG recordings showed that the number of intracellular stylet puncture and the total duration of phloem sap ingestion and concurrent salivation decreased substantially, while the total duration of non-probing increased after exposure to dimpropyridaz. Furthermore, the treatments with LC10 and LC30 of dimpropyridaz significantly reduced the longevity and fecundity of F0, and led to a decrease of the relative fitness of F0 to 0.48 and 0.32, respectively. The net reproductive rate (R0) and mean generation time (T) of F1 generation were also significantly reduced, moreover the duration of reproduction was significantly shortened. In addition, at 72 h post treatment with LC30 dimpropyridaz, the gene expression levels of JHEH and USP of cotton aphids significantly increased, while the expression of FOXO, INR, EcR and INRS decreased. These results provide basis for clarifying the toxicology of dimpropyridaz to cotton aphids, and also are beneficial for effective control of cotton aphid using dimpropyridaz.

Management of venous thromboembolism in pediatric patients: Quality assessment of clinical practice guidelines and variations in recommendations.

INTRODUCTION: We performed a quality assessment and comparison of clinical practice guidelines (CPGs) for the prevention and treatment of venous thromboembolism (VTE) in pediatric patients and to provide a clinical reference. MATERIALS AND METHODS: Electronic databases, guideline development organizations, and professional societies were searched to identify CPGs for VTE in pediatric patients between January 1, 2012, and April 7, 2022. The Appraisal of Guidelines Research & Evaluation (AGREE) II instrument was used to evaluate quality. Recommendations for preventing and treating VTE in pediatric patients were extracted via descriptive synthesis. RESULTS: Six CPGs were included. The median scores (interquartile range [IQR]) for each AGREE II domain were as follows: scope and purpose, 88.89 % (IQR: 8.33 %); stakeholder involvement, 88.89 % (25 %); rigor of development, 67.71 % (24.47 %); clarity and presentation, 88.89 % (0 %); applicability, 50 % (42.71 %); and editorial independence, 66.67 % (50.00 %). In total, 268 key recommendations were extracted, and traditional anticoagulants (heparin and warfarin) remain the standard treatment. However, in recent years direct oral anticoagulants (DOACs) have shown similar efficacy and safety results for the treatment of VTE in children to those reported in adults; therefore, this practice is recommended in recent guidelines. CONCLUSIONS: Variability exists in the development and reporting of CPGs for VTE in pediatric patients. There may be changes to the recommendations for the prevention and treatment of VTE in pediatrics in the future due to the efficacy of DOACs in children, and recommendations should be revised periodically as new evidence emerges.

Cells and Fugu Response to Capsid of BFNNV Genotype.

The nervous necrosis virus (NNV) of the BFNNV genotype is the causative agent of viral encephalopathy and retinopathy (VER) in cold water fishes. Similar to the RGNNV genotype, BFNNV is also considered a highly destructive virus. In the present study, the RNA2 of the BFNNV genotype was modified and expressed in the EPC cell line. The subcellular localization results showed that the capsid and N-terminal (1-414) were located in the nucleus, while the C-terminal (415-1014) of the capsid was located in the cytoplasm. Meanwhile, cell mortality obviously increased after expression of the capsid in EPC. EPC cells were transfected with pEGFP-CP and sampled at 12 h, 24 h and 48 h for transcriptome sequencing. There are 254, 2997 and 229 up-regulated genes and 387, 1611, and 649 down-regulated genes post-transfection, respectively. The ubiquitin-activating enzyme and ubiquitin-conjugating enzyme were up-regulated in the DEGs, indicating that cell death evoked by capsid transfection may be related to ubiquitination. The qPCR results showed that heat stock protein 70 (HSP70) is extremely up-regulated after expression of BFNNV capsid in EPC, and N-terminal is the key region to evoke the high expression. For further study, the immunoregulation of the capsid in fish pcDNA-3.1-CP was constructed and injected into the Takifugu rubripes muscle. pcDNA-3.1-CP can be detected in gills, muscle and head kidney, and lasted for more than 70 d post-injection. The transcripts of IgM and interferon inducible gene Mx were up-regulated after being immunized in different tissues, and immune factors, such as IFN-γ and C3, were also up-regulated in serum, while C4 was down-regulated one week after injection. It was suggested that pcDNA-3.1-CP can be a potential DNA vaccine in stimulating the immune system of T. rubripes; however, NNV challenge needs to be conducted in the following experiments.

Comparative Efficacy and Safety of Thrombolytic Agents for Pulmonary Embolism: A Bayesian Network Meta-Analysis.

BACKGROUND: Thrombolytic agents and anticoagulants are the two classes of medication used in the treatment of acute pulmonary embolism (PE). There is continuous renewal and iteration of thrombolytic agents, and the efficacy and adverse effects of different agents have different effects on PE due to their different mechanisms of action. OBJECTIVES: The aim of the study was to evaluate the efficacy and safety of different thrombolytic agents in the treatment of all types of acute PE: hemodynamically unstable PE (massive PE) and hemodynamically stable PE (submassive PE and low-risk PE), using a network meta-analysis. METHODS: A search was conducted of the following databases: PubMed, The Cochrane Library, Embase, and Web of Science to collect randomized controlled trials (RCTs) comparing thrombolytic agents with heparin or other thrombolytic agents in patients with acute PE; the clinical outcomes included patient mortality, recurrent PE, pulmonary artery systolic pressure (PASP) after treatment, and major and minor bleeding. The measurement duration of outcome indicators was the longest follow-up period. Thereafter, a network meta-analysis was performed using a Bayesian network framework. RESULTS: A total of 29 RCTs (3,067 patients) were included, of which 6 studies (304 patients) were massive PE, 14 studies (2,173 patients) were submassive PE, 1 study (83 patients) included massive and submassive PE, and 8 studies (507 patients) were PE of unknown type. The treatment regimens included thrombolytic therapy (alteplase, reteplase, tenecteplase, streptokinase, and urokinase) and anticoagulant therapy alone. The results showed that the mortality using thrombolytic agents (except tenecteplase) was significantly lower compared with heparin. The recurrence of PE with alteplase was significantly lower compared with heparin (RR = 0.23, 95% CI, 0.04, 0.65). The PASP after using alteplase was significantly lower compared with heparin (mean difference = -11.36, 95% CI, -21.45, -1.56). Compared with heparin, the incidence of minor bleeding associated with tenecteplase was higher (RR = 3.27, 95% CI, 1.36, 7.39); compared with streptokinase, the incidence of minor bleeding associated with tenecteplase was higher (RR = 3.22, 95% CI, 1.01, 11.10). CONCLUSION: For patients with acute PE, four thrombolytic agents (alteplase, reteplase, streptokinase, and urokinase) appeared to be superior in efficacy compared with anticoagulants alone due to a reduction in mortality and no increase in bleeding risk. Alteplase may be a better choice because it not only reduced mortality but also reduced PE recurrence rate and treated PASP. Tenecteplase did not reduce mortality compared with anticoagulants alone and may not be a good choice of thrombolytic agent due to an increase in minor bleeding compared with streptokinase and anticoagulants alone. Thrombolytic drugs should be rationally selected to optimize the thrombolytic regimen and achieve as good a balance as possible between thrombolysis and bleeding.

Analysis of the Mitogenomes of Two Helotid Species Provides New Insights into the Phylogenetic Relationship of the Basal Cucujoidea (Insecta: Coleoptera).

Helotid beetles are commonly found in places where sap flows from tree trunks and in crevices in bark. The Helotidae family is a rare and primitive group of Cucujoidea. To date, no complete mitochondrial (mt) genome has been sequenced for this family. To better understand the characteristics of the mt genome and the evolution of Cucujoidea, we sequenced and annotated the complete mt genomes of Helota thoracica (Ritsema, 1895) and Helota yehi Lee, 2017 using next-generation sequencing. These are the first record of Helotidae mt genomes. The RNA secondary structures of both species were also predicted in this study. The mt genomes of H. thoracica and H. yehi are circular, with total lengths of 16,112 bp and 16,401 bp, respectively. After comparing the mt genomes of H. thoracica and H. yehi, we observed the gene arrangement, codon usage patterns, base content, and RNA secondary structures of both species to be similar, which has also been noted in other Coleoptera insects. The nucleotide sequence of the coding regions and the control region has small differences. The phylogenetic analysis indicated that Helotidae and Protocucujidae are sister groups and revealed the relationship between seven families; however, the validity of the two series (Erotylid series and Nitidulid series) as larger groups in the superfamily was not supported. The mt phylogenomic relationships have strong statistical support. Therefore, the division of Cucujoidea into series should be re-examined. Our results will provide a better understanding of the mt genome and phylogeny of Helotidae and Cucujoidea and will provide valuable molecular markers for further genetic studies.

Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus.

Interferon γ (IFN-γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN-γ is best described in humans, but less of IFN-γ in fish species has been described at protein level. In the present study, IFN-γ from Gadus macrocephalus (GmIFN-γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN-γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN-γ signature sequence at the C-terminal. GmIFN-γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN-γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN-γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET-32a-GmIFN-γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA-GmIFN-γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN-γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1-GmIFN-γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN-γ protein could also induce MICA expression, indicating that GmIFN-γ could stimulate immune response. The yeast GS115 with GmIFN-γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN-γ was upregulated significantly; however, genes of the interferon type-I signal pathway were not well stimulated.

Management of Ventilator-Associated Pneumonia: Quality Assessment of Clinical Practice Guidelines and Variations in Recommendations on Drug Therapy for Prevention and Treatment.

Purpose: To assess the quality of clinical practice guidelines (CPGs) related to drug therapy for prevention and control of ventilator-associated pneumonia (VAP) and compare the differences and similarities between recommendations. Methods: Electronic databases (including PubMed, Cochrane library, Embase, Web of Science), guideline development organizations, and professional societies were searched to identify CPGs for VAP from 20 January 2012 to 20 January 2022. The Appraisal of Guidelines Research & Evaluation (AGREE) II instrument was used to evaluate the quality of the guidelines. The recommendations on drug therapy for prevention and treatment for each guideline were extracted, and then a descriptive synthesis was performed to analyze the scope/topic, and consistency of the recommendations. Results: Thirteen CPGs were included. The median score and interquartile range (IQR) in each domain are shown below: scope and purpose 72.22% (63.89%,83.33%); stakeholder involvement 44.44% (38.89%,52.78%); rigor of development 43.75% (31.25%,57.29%); clarity and presentation 94.44% (77.78%,94.44%); applicability 20.83 (8.34%,33.34%) and editorial independence 50% (33.33%,66.67%). We extracted 21 recommendations on drug therapy for prevention of VAP and 51 recommendations on drugs used for treatment. Some controversies remained among the included guidelines. Conclusion: There is considerable variability in the development processes and reporting of VAP guidelines. Despite many similarities, the recommendations still had some inconsistencies in the details. For the prevention and treatment of VAP, local microbial epidemiology and antibiotic sensitivity must be considered, and recommendations should be regularly revised as new evidence emerges.

Characteristics and main factors of foam flow in broken rock mass in coal mine goaf.

To protect the environment and reduce the occurrence of coal mine fire, foam injection in goafs is an effective measure for preventing and extinguishing mine fires. The flow characteristics of foams injected into goafs have a significant impact on the prevention and extinguishment of such fires. To study the flow characteristics of foam injected into a goaf, we first independently constructed a set of experimental platforms for the visualization of goafs. Next, we performed physical experiments on foam injection using similarity theory. Flow characteristics were simulated under different foam concentrations, flow rates, and goaf porosities. The exponential function was found to provide a good fit to the trajectory of the foam's stacking edge in the goaf. According to the foam injection volume, the trend of the fitting equation parameter a could be divided into two stages. The first stage was the rapidly decreasing stage, and the second stage was the stable stage. It was inferred that the stacking height and diffusion radius of the foam under different conditions were related to the speed of liquid film drainage. The results of this study can provide a valuable reference for the use of fire prevention and extinguishment technology in the goaf.

Modeling drug combination effects via latent tensor reconstruction.

MOTIVATION: Combination therapies have emerged as a powerful treatment modality to overcome drug resistance and improve treatment efficacy. However, the number of possible drug combinations increases very rapidly with the number of individual drugs in consideration, which makes the comprehensive experimental screening infeasible in practice. Machine-learning models offer time- and cost-efficient means to aid this process by prioritizing the most effective drug combinations for further pre-clinical and clinical validation. However, the complexity of the underlying interaction patterns across multiple drug doses and in different cellular contexts poses challenges to the predictive modeling of drug combination effects. RESULTS: We introduce comboLTR, highly time-efficient method for learning complex, non-linear target functions for describing the responses of therapeutic agent combinations in various doses and cancer cell-contexts. The method is based on a polynomial regression via powerful latent tensor reconstruction. It uses a combination of recommender system-style features indexing the data tensor of response values in different contexts, and chemical and multi-omics features as inputs. We demonstrate that comboLTR outperforms state-of-the-art methods in terms of predictive performance and running time, and produces highly accurate results even in the challenging and practical inference scenario where full dose-response matrices are predicted for completely new drug combinations with no available combination and monotherapy response measurements in any training cell line. AVAILABILITY AND IMPLEMENTATION: comboLTR code is available at https://github.com/aalto-ics-kepaco/ComboLTR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The evaluation of a nurse-led hypertension management model in an urban community healthcare: A randomized controlled trial.

BACKGROUND: Hypertension is a silent disease of the masses with an increasing prevalence and poor control rates. This study aims to establish and test the efficacy of a nurse-led hypertension management model in the community. METHODS: A single-blind, randomized controlled trial was performed. 156 hypertensive patients with uncontrolled blood pressure were equally and randomly allocated into 2 groups. Patients in the study group received a 12-week period of hypertension management. Blood pressure, self-care behaviors, self-efficacy, and satisfaction were assessed at the start of recruitment, 12 and 16 weeks thereafter. RESULTS: After the intervention, blood pressure of patients in the study group had greater improvement in self-care behaviors and a higher level of satisfaction with the hypertensive care compared to the control group (both P < .05). CONCLUSIONS: The nurse-led hypertension management model is feasible and effective for patients with uncontrolled blood pressure in the community.

Synthesis and biological evaluation of 6-phenylpurine linked hydroxamates as novel histone deacetylase inhibitors.

A series of 6-phenylpurine based hydroxamates have been designed, synthesized and evaluated. Compound 3b and its analogs are potent histone deacetylase (HDAC) but weak PI3K/mTOR inhibitors. These compounds demonstrated broad anti-cancer activities against 38 cancer cell lines with leukemia, lymphoma, and the majority of liver cancer cell lines exhibiting the most sensitivity towards these compounds. Compound 3b demonstrated modulation of HDAC targets in vitro in a dose-dependent manner. It has good in vitro ADME profile that translated into a greatly improved pharmacokinetic profile. 3b also demonstrated modulation of HDACs in tumors in a PC-3 xenograft model. It was further evaluated in combination therapies in vitro. It exhibited additive or synergistic growth inhibition effect in HepG2 cells when combined with a number of approved drugs such as sorafenib, sunitinib, and erlotinib. Hence, 3b has the potential to be combined with the above to treat advanced liver cancer. As such, current data warrant further evaluation, optimization, and subsequent in vivo validation of the potential combination therapies.

Sugar transporter genes in grass carp (Ctenopharyngodon idellus): molecular cloning, characterization, and expression in response to different stocking densities.

Glucose and fructose play a central role in the metabolism and cellular homeostasis of organisms. Their absorption is co-mediated by two families of glucose transporters, Na+-coupled glucose co-transporters (SGLTs) and facilitative Na+-independent sugar carriers (GLUTs), in the intestine. However, limited information has been available on these transporters in fish. Therefore, we studied glut2, sglt1, and sglt4 genes in grass carp (Ctenopharyngodon idellus). The full-length cDNAs of glut2 was 2308 bp, with an open reading frame (ORF) of 503 amino acids (AAs). The full-length cDNAs of sglt1 was 2890 bp, with an ORF of 658 AAs. Additionally, the full-length cDNAs of sglt4 was 2090 bp, with an ORF encoding 659 AAs. The three deduced AA sequences showed high homology between grass carp and other cyprinid fish species. Based on homology modeling, three-dimensional models of GLUT2, SGLT1, and SGLT4 proteins were created and transmembrane domains were noted. glut2, sglt1, and sglt4 were abundantly expressed in the anterior and mid intestine. In particular, glut2 was markedly expressed in liver (P < 0.05). Additionally, the results indicated that different stocking densities (0.9 or 5.9 kg m-2) did not alter intestinal section-dependent expression patterns of the three transporter genes. However, high stocking density impacted segmental mRNA expression levels. This work demonstrated that mRNA expression of sugar transporter genes in the fish intestine was segment specific, and crowding stress may affect the activity of intestinal sugar transporters. These results provided new insights into the relationship between crowding stress and intestinal sugar transporters in fish.

Antibody neutralization of microbiota-derived circulating peptidoglycan dampens inflammation and ameliorates autoimmunity.

The human microbiota provides tonic signals that calibrate the host immune response1,2, but their identity is unknown. Bacterial peptidoglycan (PGN) subunits are likely candidates since they are well-known immunity-enhancing adjuvants, released by most bacteria during growth, and have been found in the blood of healthy people3-7. We developed a monoclonal antibody (mAb), 2E7, that targets muramyl-L-alanyl-D-isoglutamine (MDP), a conserved and minimal immunostimulatory structure of PGN. Using 2E7-based assays, we detected PGN ubiquitously in human blood at a broad range of concentrations that is relatively stable in each individual. We also detected PGN in the serum of several warm-blooded animals. However, PGN is barely detectable in the serum of germ-free mice, indicating that its origin is the host microbiota. Neutralization of circulating PGN via intraperitoneal administration of 2E7 suppressed the development of autoimmune arthritis and experimental autoimmune encephalomyelitis in mice. Arthritic NOD2-/- mice lacking the MDP sensor did not respond to 2E7, indicating that 2E7 dampens inflammation by blocking nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-mediated pathways. We propose that circulating PGN acts as a natural immune potentiator that tunes the host immune response; altering its level is a promising therapeutic strategy for immune-mediated diseases.

Design, Synthesis, and Preclinical Evaluation of Fused Pyrimidine-Based Hydroxamates for the Treatment of Hepatocellular Carcinoma.

Class I histone deacetylases (HDACs) are highly expressed and/or upregulated in hepatocellular carcinoma (HCC) and are associated with aggressiveness, spread, and increased mortality of HCC. Activation of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway was involved in the development of HCC and acquired resistance to sorafenib. A series of purine or 5H-pyrrolo[3,2-d]pyrimidine based hydroxamates were designed and developed as multitarget drugs to modulate both HDACs and the PI3K/Akt/mTOR pathway. Among 39 cell lines screened, the molecules (e.g., 20e, 20f, and 20q) were the most selective against leukemia, lymphoma, and HCC cells; they also demonstrated target modulation in cancer cell lines and in mice bearing MV4-11 and HepG2 tumors. Compound 20f in particular showed significant single agent oral efficacy in hypervascular liver cancer models (e.g., HepG2, HuH-7, and Hep3B) and was well-tolerated. These encouraging results, along with its favorable target profile and tissue distribution, warrant further development of 20f.

Aquaporins: Novel Targets for Age-Related Ocular Disorders.

Aquaporins (AQPs), a large family of membrane protein channels that facilitate transport of water and other small solutes, play important roles in physiological functions and human diseases. Up till now, 13 types of AQPs, numbered 0 through 12, have been identified in various mammalian tissues. Homologous genes for AQPs in amphibians, insects, and bacteria highlight the evolutionary conservation and, thus, the importance of these membrane channels. Many members of the AQP family are expressed in the eye. AQP1, which is a water-selective channel, is expressed in the anterior chamber (cornea, ciliary body, trabecular meshwork) and posterior chamber (retina and microvessels in choroid), controlling the fluid homeostasis in the eye. Mice knockout studies have indicated that AQP1 plays an important function in the eye by suggesting its role in aqueous humor dynamics and retina angiogenesis. This review will focus on the role of AQP1 as a novel target for ocular disorders such as glaucoma and age-related macular degeneration, and it will discuss challenges and advances in identifying modulators of AQP1 function that could be useful in clinical applications.

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs.

Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA of Haliotis discus hannai from Fujian, China.

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)3 displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.

Structure and ligand-based design of mTOR and PI3-kinase inhibitors leading to the clinical candidates VS-5584 (SB2343) and SB2602.

Phosphoinositide 3-kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) act as critical effectors in a commonly deregulated cell signaling pathway in human cancers. The abnormal activation of the PI3K/mTOR pathway has been shown to play a role in initiation, progression, and metastasis of human tumors. Being one of the most frequently activated pathways in cancer, much effort has been directed toward inhibition of the PI3K/mTOR pathway as a novel oncology therapy. Previous work by a number of groups has revealed several selective PI3K and dual mTOR/PI3K inhibitors. However, there are few reports of therapeutic agents with a pan-PI3K/mTOR inhibitory profile within a narrow concentration range. We therefore initiated a drug discovery project with the aim of discovering dual mTOR/PI3K inhibitors which would equipotently inhibit the 4 isoforms of PI3K, α, ß, γ, and δ, and mTOR a compelling profile for powerful blockage of the PI3K/mTOR pathway. A pharmacophore model was generated and used for designing a series of novel compounds, based on a purine scaffold, which potently inhibited mTOR and PI3Ks. These compounds contained a phenol headgroup essential for binding to the target proteins. Early efforts concentrated on finding replacements for the phenol as it was rapidly conjugated resulting in a short half-life in vivo. Compounds with a variety of headgroups were docked into the PI3Kα and mTOR ATP-binding sites, and aminopyrimidine and aminopyrazine were found to make excellent phenol replacements. Further structure guided optimization of side chains in the 8- and 9-positions of the purine resulted in potent inhibitors with good PKDM properties. As the PI3 kinases play a role in insulin signaling, it is believed that targeting mTOR selectively may give the benefit of blocking the AKT-pathway while avoiding the potential side effects associated with PI3K inhibition. As a result we designed a further series of selective mTOR kinase inhibitors. The project was successfully concluded by progressing both a dual mTOR/PI3K inhibitor, SB2343, and a selective mTOR inhibitor, SB2602, into preclinical development. SB2343 has since entered phase 1 clinical development as VS-5584.