“Snapshots” of Ispinesib-induced Conformational Changes in the Mitotic Kinesin Eg5

Kinesins comprise a superfamily of molecular motors that drive a wide variety of cellular physiologies, from cytoplasmic transport to formation of the bipolar spindle in mitosis. These differing roles are reflected in corresponding polymorphisms in key kinesin structural elements. One of these is a unique loop and stem motif found in all kinesins and referred to as loop 5 (L5). This loop is longest in the mitotic kinesin Eg5 and is the target for a number of small molecule inhibitors, including ispinesib, which is being used in clinical trials in patients with cancer. In this study, we have used x-ray crystallography to identify a new structure of an Eg5-ispinesib complex and have combined this with transient state kinetics to identify a plausible sequence of conformational changes that occur in response to ispinesib binding. Our results demonstrate that ispinesib-induced structural changes in L5 from Eg5 lead to subsequent changes in the conformation of the switch II loop and helix and in the neck linker. We conclude that L5 in Eg5 simultaneously regulates the structure of both the ATP binding site and the motor''s mechanical elements that generate force.     

Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells

Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.     

Fine‐scale vertical and horizontal movements of juvenile yellowfin tuna (Thunnus albacares) associated with a subsurface fish aggregating device (FAD) off southwestern Taiwan

An increase in yellowfin tuna (Thunnus albacares) catch by danish seine fisheries around the subsurface fish aggregating devices (FADs) in southern Taiwan waters has been a concern of local government and environmental groups. However, the attraction mechanism of aggregating tunas at the subsurface FADs is still poorly understood. The objective of this study is to examine the fine‐scale vertical and horizontal movements of juvenile yellowfin tunas around a subsurface FAD. In total, 53 tunas (35–81 cm fork length) were tagged with ultrasonic telemetry tags and released at a subsurface FAD in the waters off Shiao‐Liu‐Chiu Island, southwestern Taiwan from October 2008 to December 2009. These tunas stayed at the subsurface FAD for up to 31 days, with daytime vertical movement depths averaging 60–80 m at a maximum depth of 250 m. At night, the tuna gathered at a shallow depth of 40 m. The mean depth of vertical movement in the daytime is significantly different from that of the nighttime (P < 0.05). The maximum detectable distance of horizontal movement was 1.6 km, with 80% of the long horizontal movements occurring in the daytime. It is likely that the purpose of these vertical and horizontal movements was for feeding or avoiding predators. Moreover, the tagged tunas did not depart from the subsurface FAD simultaneously, suggesting distinct behaviors in their movements.     

MicroRNA-33a functions as a bone metastasis suppressor in lung cancer by targeting parathyroid hormone related protein

Background

Bone is a common site of metastasis for lung cancer, and is associated with significant morbidity and a dismal prognosis. MicroRNAs (miRNAs) are increasingly implicated in regulating the progression of malignancies.

Methods

The efficacy of miR-33a or anti-miR-33a plasmid was assessed by Real-time PCR. Luciferase assays were using One-Glo Luciferase Assay System. Measurement of secreted factors was determined by ELISA kit.

Results

We have found that miR-33a, which is downregulated in lung cancer cells, directly targets PTHrP (parathyroid hormone-related protein), a potent stimulator of osteoclastic bone resorption, leading to decreased osteolytic bone metastasis. We also found that miR-33a levels are inversely correlated with PTHrP expression between human normal bronchial cell line and lung cancer cell lines. The reintroduction of miR-33a reduces the stimulatory effect of A549 on the production of osteoclastogenesis activator RANKL (receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) on osteoblasts, while the expression of PTHrP is decreased in A549 cells. miR-33a overexpression also reduces the inhibitory activity of A549 on the production of OPG (osteoprotegerin), an osteoclastogenesis inhibitor. In addition, miR-33a-mediated PTHrP downregulation results in decreased IL-8 secretion in A549, which contributes to decreased lung cancer-mediated osteoclast differentiation and bone resorption.

Conclusions

These findings have led us to conclude that miR-33a may be a potent tumor suppressor, which inhibits direct and indirect osteoclastogenesis through repression of PTHrP.

General significance

miR-33a may even predict a poor prognosis for lung cancer patients.     

Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8⁺ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8⁺ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8⁺ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic antitumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8⁺ T cells, which led to higher ratios of CD8⁺/Treg and CD8⁺/CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8⁺ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells.     

Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.     

Fibromatosis stem cells rather than bone-marrow mesenchymal stem cells recapitulate a murine model of fibromatosis

Palmar fibromatosis is a benign fibroproliferative tumor of unknown etiology, with a high rate of recurrence after excision. The offending cells of palmar fibromatosis are myofibroblasts and the cellular origin of other myofibroblasts has previously been reported to be the bone marrow. However, further clarification of the relationship between bone marrow precursors and palmar fibromatosis is required. Stem cells (SCs) are known to exist in various tissues, but whether SCs can be isolated from fibromatosis tissue is still unclear. The purpose of this study was to isolate and identify stem cells from human palmar fibromatosis, and to evaluate the differences in the differentiation and fibrogenic capacities of bone marrow stem cells (BMSCs) and fibromatosis-derived stem cells (FSCs). We found that FSCs had better fibrogenic differentiation potential than BMSCs, whereas BMSCs had better adipogenic and chondrogenic differentiation capacities. Treatment with transforming growth factor-β1 increased the expression of α-smooth muscle actin, and types III and I collagen significantly more in FSCs than in BMSCs. An in vivo study further confirmed the results of fibrogenesis and suggested that FSCs can recapitulate the fibromatosis nodule. In summary, their myofibroblastic differentiation both in vivo and in vitro makes FSCs a potential cell source for future applications in murine models of fibromatosis or fibrogenesis.     

Population genetic structure of the newly invasive Q biotype of Bemisia tabaci in Taiwan

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is among the top 100 invasive pests in the world, and it causes serious agricultural damage and economic losses in many countries. More than 24 biotypes of the sweetpotato whitefly have been detected worldwide, of which the Q biotype has recently been reported to be a new invasive pest spreading throughout the world via trade in poinsettias, Euphorbia pulcherrima Willd. ex Klotzsch (Euphorbiaceae). In 2006, the Q biotype was first recorded in Taiwan in greenhouses, but not in the field, suggesting that the invasion of this biotype might be at an early stage in that country. The mitochondrial cytochrome oxidase I (COI) gene and 12 microsatellite loci were used to investigate the genetic structure of multiple B. tabaci Q biotype populations. The presence of only a few COI haplotypes and a low number of nucleotide differences suggest high genetic similarity among these populations. Microsatellite analyses also revealed low genetic differentiation and frequent gene flow among greenhouses. The molecular evidence supports the occurrence of a recent genetic bottleneck in the B. tabaci Q biotype. Bayesian cluster analyses indicated that at least two invasion events have occurred in Taiwan. Phylogenetic analyses of microsatellites support Q biotype migration among greenhouses, which was likely facilitated by the frequent movement of poinsettias between greenhouses. Future management strategies should focus on developing plantlet trade regulations to avoid further anthropogenic dispersal of the B. tabaci Q biotype among greenhouses in Taiwan.     

Targeting lipid droplet lysophosphatidylcholine for cisplatin chemotherapy

This study aims to explore lipidic mechanism towards low‐density lipoprotein receptor (LDLR)‐mediated platinum chemotherapy resistance. By using the lipid profiling technology, LDLR knockdown was found to increase lysosomal lipids and decrease membranous lipid levels in EOC cells. LDLR knockdown also down‐regulated ether‐linked phosphatidylethanolamine (PE‐O, lysosomes or peroxisomes) and up‐regulated lysophosphatidylcholine [LPC, lipid droplet (LD)]. This implies that the manner of using Lands cycle (conversion of lysophospholipids) for LDs might affect cisplatin sensitivity. The bioinformatics analyses illustrated that LDLR‐related lipid entry into LD, rather than an endogenous lipid resource (eg Kennedy pathway), controls the EOC prognosis of platinum chemotherapy patients. Moreover, LDLR knockdown increased the number of platinum‐DNA adducts and reduced the LD platinum amount. By using a manufactured LPC‐liposome‐cisplatin (LLC) drug, the number of platinum‐DNA adducts increased significantly in LLC‐treated insensitive cells. Moreover, the cisplatin content in LDs increased upon LLC treatment. Furthermore, lipid profiles of 22 carcinoma cells with differential cisplatin sensitivity (9 sensitive vs 13 insensitive) were acquired. These profiles revealed low storage lipid levels in insensitive cells. This result recommends that LD lipidome might be a common pathway in multiple cancers for platinum sensitivity in EOC. Finally, LLC suppressed both cisplatin‐insensitive human carcinoma cell training and testing sets. Thus, LDLR‐platinum insensitivity can be due to a defective Lands cycle that hinders LPC production in LDs. Using lipidome assessment with the newly formulated LLC can be a promising cancer chemotherapy method.     

Arabidopsis thaliana as a model system for testing the effect of Trichoderma volatile organic compounds

In ecosystems, plant and bacterial volatile organic compounds (VOCs) are known to influence plant growth but less is known about the physiological effects of fungal VOCs. We have used Arabidopsis thaliana as a model to test the effects of VOCs from the soil fungus Trichoderma viride. Mature colonies of T. viride cultured on Petri plates were placed in a growth chamber in a shared atmosphere with A. thaliana without direct physical contact. Compared to controls, plants grown in the presence of T. viride volatiles were taller, bigger, flowered earlier, and had more lateral roots. They also had increased total biomass (45 %) and chlorophyll concentration (58 %). GC–MS analysis of T. viride VOCs revealed 51 compounds of which isobutyl alcohol, isopentyl alcohol, and 3-methylbutanal were most abundant. We conclude that VOCs emitted by T. viride have growth promoting effects on A. thaliana in the absence of direct physical contact.