Design and Synthesis of Fluoroacylshikonin as an Anticancer Agent

A series of shikonin derivatives, selectively acylated by various fluorinated carboxylic acids at the side chain of shikonin, were synthesized and their anticancer activity evaluated, in which eight compounds are reported for the first time. Among all the compounds tested, compound S7 showed the most potent anticancer activity against B16‐F10 (malignant melanoma cells), MG63 (human osteosarcoma cells), and A549 (lung cancer cells) with IC₅₀ 0.39 ± 0.01, 0.72 ± 0.04 and 0.58 ± 0.02 µmol/L. Docking simulation of compound S7 was carried out to position S7 into a tubulin active site to determine the probable binding conformation. All the results suggested that compound S7 may be a potential anticancer agent. Chirality 25:757–762, 2013. © 2013 Wiley Periodicals, Inc.     

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2α on human umbilical cord vessels

The effect of six naturally occurring prostaglandins on isolated umbilical arteries and veins has been studied. All six prostaglandins had a constricting effect on the umbilical vessels. On the umbilical artery preparations the potencies in decreasing order were A₂>B₂>F_2α>B₁>E₂>A₁. Prostaglandin B₂ was more potent than PGA₂ on the umbilical vein. Polyphloretin phosphate (PPP) antagonised the constricting effect of all six prostaglandins without altering responses to 5-hydroxytryptamine.     

Protective Role of False Tendon in Subjects with Left Bundle Branch Block: A Virtual Population Study

False tendons (FTs) are fibrous or fibromuscular bands that can be found in both the normal and abnormal human heart in various anatomical forms depending on their attachment points, tissue types, and geometrical properties. While FTs are widely considered to affect the function of the heart, their specific roles remain largely unclear and unexplored. In this paper, we present an in silico study of the ventricular activation time of the human heart in the presence of FTs. This study presents the first computational model of the human heart that includes a FT, Purkinje network, and papillary muscles. Based on this model, we perform simulations to investigate the effect of different types of FTs on hearts with the electrical conduction abnormality of a left bundle branch block (LBBB). We employ a virtual population of 70 human hearts derived from a statistical atlas, and run a total of 560 simulations to assess ventricular activation time with different FT configurations. The obtained results indicate that, in the presence of a LBBB, the FT reduces the total activation time that is abnormally augmented due to a branch block, to such an extent that surgical implant of cardiac resynchronisation devices might not be recommended by international guidelines. Specifically, the simulation results show that FTs reduce the QRS duration at least 10 ms in 80% of hearts, and up to 45 ms for FTs connecting to the ventricular free wall, suggesting a significant reduction of cardiovascular mortality risk. In further simulation studies we show the reduction in the QRS duration is more sensitive to the shape of the heart then the size of the heart or the exact location of the FT. Finally, the model suggests that FTs may contribute to reducing the activation time difference between the left and right ventricles from 12 ms to 4 ms. We conclude that FTs may provide an alternative conduction pathway that compensates for the propagation delay caused by the LBBB. Further investigation is needed to quantify the clinical impact of FTs on cardiovascular mortality risk.     

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.     

A dominant-negative approach that prevents diphthamide formation confers resistance to Pseudomonas exotoxin A and diphtheria toxin

Diphtheria toxin (DT), Pseudomonas aeruginosa Exotoxin A (ETA) and cholix toxin from Vibrio cholerae share the same mechanism of toxicity; these enzymes ADP-rybosylate elongation factor-2 (EF-2) on a modified histidine residue called diphthamide, leading to a block in protein synthesis. Mutant Chinese hamster ovary cells that are defective in the formation of diphthamide have no distinct phenotype except their resistance to DT and ETA. These observations led us to predict that a strategy that prevents the formation of diphthamide to confer DT and ETA resistance is likely to be safe. It is well documented that Dph1 and Dph2 are involved in the first biochemical step of diphthamide formation and that these two proteins interact with each other. We hypothesized that we could block diphthamide formation with a dominant negative mutant of either Dph1 or Dph2. We report in this study the first cellular-targeted strategy that protects against DT and ETA toxicity. We have generated Dph2(C-), a dominant-negative mutant of Dph2, that could block very efficiently the formation of diphthamide. Cells expressing Dph2(C-) were 1000-fold more resistant to DT than parental cells, and a similar protection against Pseudomonas exotoxin A was also obtained. The targeting of a cellular component with this approach should have a reduced risk of generating resistance as it is commonly seen with antibiotic treatments.     

A hypomorphic mutation in Lpin1 induces progressively improving neuropathy and lipodystrophy in the rat

The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critical role in lipid metabolism. In this study we describe the identification and characterization of a rat model with a mutated Lpin1 gene (Lpin1(1Hubr)), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin1(1Hubr) rats are characterized by hindlimb paralysis and mild lipodystrophy that are detectable from the second postnatal week. Sequencing of Lpin1 identified a point mutation in the 5'-end splice site of intron 18 resulting in mis-splicing, a reading frameshift, and a premature stop codon. As this mutation does not induce nonsense-mediated decay, it allows the production of a truncated Lipin 1 protein lacking PAP1 activity. Lpin1(1Hubr) rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1(fld/fld) mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular analyses revealed that these lesions improve in older Lpin1(1Hubr) rats as compared with young Lpin1(1Hubr) rats and Lpin1(fld/fld) mice. We observed activation of compensatory biochemical pathways substituting for missing PAP1 activity that, in combination with a possible non-enzymatic Lipin 1 function residing outside of its PAP1 domain, may contribute to the less severe phenotypes observed in Lpin1(1Hubr) rats as compared with Lpin1(fld/fld) mice. Although we are cautious in making a direct parallel between the presented rodent model and human disease, our data may provide new insight into the pathogenicity of recently identified human LPIN1 mutations.     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998     

Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation

Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) α/β, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKCα/β and δ inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in β3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.Platelet activation is initiated through several classes of membrane receptors, which are stimulated by agonists produced at the vascular lesion (1–3). A second wave of signaling, caused by engagement of integrins, occurs as platelets bind to the lesion surface and aggregate (4). Together, these plasma membrane proteins initiate the platelet processes important for thrombosis (e.g. adhesion, spreading, secretion, and clot retraction). Small GTP-binding proteins, specifically members of the Ras superfamily, link signaling events from various platelet receptors to defined outcomes, such as shape change (5–7), aggregation (8, 9), and secretion (10–12). Rab proteins play roles in granule secretion, with Rab4 and Rab6 being involved in alpha granule release (10, 11) and Rab27a/b in dense core granule release (12, 13). RalA is activated in response to various stimuli (14–16) and may play a role in secretion by anchoring the exocyst complex to specific membrane sites (17). Rap1 plays a role in integrin α_IIbβ₃ activation (8, 9). Rho family GTPases (Rho, Rac, and Cdc42) play roles in platelet phosphoinositide signaling and in the regulation of the actin cytoskeleton (5–7). While these small GTP-binding proteins are clearly important to platelet function, it is equally clear that other small G proteins are present and functional in platelets (18).The ADP-ribosylation factor (Arf)² family are Ras-related, small GTPases that affect both vesicular transport and cytoskeletal dynamics (19, 20). Based on their primary sequences, this family is divided into three classes, with Arf6 as the only member of class III (19). Arf6-GTP is considered the “active state” and can interact with downstream effectors, such as phospholipase D (PLD) (21), phosphatidylinositol 4-phosphate 5-kinase type α (22), and arfaptin 2 (23, 24), resulting in the recruitment of these effectors to the plasma membrane. The Arf6 GTP/GDP cycle is mediated by interactions with guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The large number of Arf-GEF and -GAP proteins have been discussed in recent reviews where it was noted that, unlike other small GTPases, Arf functions are generally not mediated solely by the GTP-bound state but through its cycling between states (19, 20, 25, 26).The effects that Arf6 has on the secretion and actin dynamics in nucleated cells make it an ideal candidate for function in platelets. Arf6 influences cortical actin and is important for spreading, ruffling, migration, and phagocytosis (reviewed in Ref. 19). Our previous work (27) showed that Arf6 is present on platelet membranes and is important for platelet function. Unlike other small G proteins, the Arf6 GTP-bound form is readily detectible in resting platelets and upon activation with collagen or convulxin there is a rapid conversion to the GDP-bound form. Acylated peptides, which mimic the myristoylated N terminus of Arfs have been used as isoform-specific inhibitors (28). In platelets, a myristoylated-Arf6 (myr-Arf6) peptide specifically blocks the activation-dependent loss of Arf6-GTP. This peptide also blocks aggregation, spreading on collagen, and activation of the Rho family of GTPases. Other GTPases, such as Ral and Rap, were unaffected. The simplest explanation for these data is that platelet activation stimulates the GTPase activity of Arf6, perhaps through activation of an Arf6-GAP. Alternatively, platelet activation could affect an Arf6-GEF thus reducing the production of Arf6-GTP. Regardless of mechanism, disruption of the activation-dependent loss of Arf6-GTP, with the myr-Arf6 peptide, profoundly affects the actin-based cytoskeletal rearrangements associated with platelet activation. While our initial report (27) established a role for Arf6 in platelet function, it was not clear what platelet signaling events were required to induce the loss of Arf6-GTP.In this article, we delineate the signaling cascades required for the activation-dependent loss of Arf6-GTP. We show that the Arf6-GTP to -GDP conversion was stimulated by primary agonists (thrombin, TRAP, collagen, or convulxin) but not by ADP or U46619. The decrease in Arf6-GTP, downstream of thrombin and convulxin, required PLC, and PKC activity. Loss of Arf6-GTP, via stimulation of GPVI with convulxin, additionally required Syk and PI3K activities. Pretreatment with passivators, nitric oxide (NO), and prostaglandin I₂ (PGI₂) blocked thrombin- and convulxin-induced loss of Arf6-GTP. Further experiments suggested a role for “outside-in” signaling, especially once platelet aggregates begin to form. Inclusion of RGDS peptide, EGTA, or the deletion of the β3 integrin had only minimal effects on the initial loss of Arf6-GTP but led to the partial recovery of Arf6-GTP levels. This biphasic change in Arf6-GTP levels was not seen when aggregation was allowed to occur normally. Taken together, these data show that the Arf6 nucleotide state is responsive to both initial agonist-mediated signaling and to a second wave of integrin-mediated signaling that occurs upon aggregation.